Production of dha and other lc pufas in plants

ABSTRACT

The invention provides recombinant host organisms genetically modified with a polyunsaturated fatty acid (PUFA) synthase system and one or more accessory proteins that allow for and/or improve the production of PUFAs in the host organism. The present invention also relates to methods of making and using such organisms as well as products obtained from such organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/235,435 filed Apr. 4, 2014, which claims the benefit of InternationalPatent Application PCT/US2012/048355, filed Jul. 26, 2012, designatingthe United States of America and published in English as InternationalPatent Publication WO 2013/016546 A2 on Jan. 31, 2013, which claims thebenefit under Article 8 of the Patent Cooperation Treaty and under 35U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No.61/511,878, filed Jul. 26, 2011, the disclosure of each of which ishereby incorporated herein in its entirety by this reference.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. § 1.821(c) or (e), files containing a TXT versionand a PDF version of the Sequence Listing have been submitted, titled70967-US-PCN2_211101_SequenceListing.txt, created Nov. 1, 2021 and 1,757kb in size, the contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention generally relates to recombinant host organisms(e.g., plants) genetically modified with a polyunsaturated fatty acid(PUFA) synthase system and one or more accessory proteins that allow forand/or improve the production of PUFAs in the host organism. The presentinvention also relates to methods of making and using such organisms(e.g., to obtain PUFAs) as well as products obtained from such organisms(e.g., oil and seed).

Background Art

Polyunsaturated fatty acids (PUFAs) are considered to be useful fornutritional applications, pharmaceutical applications, industrialapplications, and other purposes. However, the current supply of PUFAsfrom natural sources (e.g., fish oils) and from chemical synthesis isnot sufficient for long-term commercial needs.

Vegetable oils derived from plants (e.g., oil seed crops) are relativelyinexpensive and do not have the contamination issues associated withfish oils. However, the PUFAs found in commercially-developed plants andplant oils do not typically include more saturated or longer-chainPUFAs, and only typically include fatty acids such as linoleic acid(eighteen carbons with 2 double bonds, in the delta 9 and 12positions—18:2 delta 9,12) and linolenic acid (18:3 delta 9,12,15).

The production of more unsaturated or longer-chain PUFAs in plants bythe modification of the fatty acids endogenously produced by plants hasbeen described. For example, the genetic modification of plants withvarious individual genes encoding fatty acid elongases and/ordesaturases has been described as resulting in the generation of leavesor seeds containing significant levels of longer-chain and moreunsaturated PUFAs such as eicosapentaenoic acid (EPA), but alsocontaining significant levels of mixed shorter-chain and lessunsaturated PUFAs (Qi et al., Nature Biotech. 22:739 (2004); WO04/071467; Abbadi et al., Plant Cell 16:1 (2004); Napier and Sayanova,Proceedings of the Nutrition Society 64:387-393 (2005); Robert et al.,Functional Plant Biology 32:473-479 (2005); U.S. Appl. Pub. No.2004/0172682, U.S. Appl. No. 61/345,537, filed May 17, 2010).

Fabaceae (or Leguminosae) is a large and economically important familyof flowering plants, which is commonly known as the legume family, peafamily, bean family or pulse family. Glycine is a genus in the familyFabaceae and includes, for example, Glycine albicans, Glycine aphyonota,Glycine arenari, Glycine argyrea, Glycine canescens, Glycineclandestine, Glycine curvata, Glycine cyrtoloba, Glycine falcate,Glycine gracei, Glycine hirticaulis, Glycine hirticaulis subsp. leptosa,Glycine lactovirens, Glycine latifolia, Glycine latrobeana, Glycinemicrophylla, Glycine montis-douglas, Glycine peratosa, Glycinepescadrensis, Glycine pindanica, Glycine pullenii, Glycine rubiginosa,Glycine stenophita, Glycine syndetika, Glycine tabacina, Glycinetomentella, Glycine soja, and Glycine max (soybean). The family Fabaceaealso includes peanut, beans (Phaseolus vulgaris), broad beans (Viciafaba) or peas (Pisum sativum).

The majority of soybean oil is in the form of vegetable oils producedfor human consumption. There is also a growing market for the use ofsoybean oil in industrial applications.

BRIEF SUMMARY OF THE INVENTION

There is a need in the art for a relatively inexpensive method toefficiently and effectively produce quantities (e.g., commercialquantities) of longer-chain or more unsaturated PUFAs in plants, plantseed or plant oil, as well as quantities of lipids (e.g.,triacylglycerol (TAG) and phospholipid (PL)) enriched in such PUFAs inplants, plant seed or plant oil. A system for providing and improvingPUFA production in host organisms (e.g., plants) by providingrecombinant host organisms genetically modified with a polyunsaturatedfatty acid (PUFA) synthase and one or more accessory proteins, asdescribed herein, is a significant alternative to the approaches in theart.

The present invention is directed to genetically modified plants (e.g.,plants of the family Fabaceae or the genus Glycine such as soybean),descendants, seeds, cells, tissues, or parts thereof, comprising (i) anucleic acid sequence encoding a polyunsaturated fatty acid (PUFA)synthase (e.g., an algal PUFA synthase) that produces at least one PUFA;and (ii) a nucleic acid sequence encoding a phosphopantetheinyltransferase (PPTase) that transfers a phosphopantetheinyl cofactor to anPUFA synthase system (e.g., an algal PUFA synthase system) ACP domain.

In some embodiments of the present invention, the PUFA synthasecomprises an amino acid sequence that is 80% to 99% identical to theamino acid sequence of SEQ ID NO: 1 or comprises the amino acid sequenceof SEQ ID NO:1. In some embodiments, the nucleic acid sequence encodingthe PUFA synthase comprises a nucleic acid sequence 80% to 99% identicalto the nucleic acid sequence of SEQ ID NO:6 or comprises the nucleicacid sequence of SEQ ID NO:6. In some embodiments, the PUFA synthasecomprises an amino acid sequence that is 80% to 99% identical to theamino acid sequence of SEQ ID NO:2 or comprises the amino acid sequenceof SEQ ID NO:2. In some embodiments, the nucleic acid sequence encodingthe PUFA synthase comprises a nucleic acid sequence that is 80% to 99%identical to the nucleic acid sequence of SEQ ID NO:7 or comprises thenucleic acid sequence of SEQ ID NO:7. In some embodiments, the PUFAsynthase comprises an amino acid sequence that is 80% to 99% identicalto the amino acid sequence of SEQ ID NO:3 or comprises the amino acidsequence of SEQ ID NO:3. In some embodiments, the nucleic acid sequenceencoding the PUFA synthase comprises a nucleic acid sequence that is 80%to 99% identical to the nucleic acid sequence of SEQ ID NO:8 orcomprises the nucleic acid sequence of SEQ ID NO:8. In some embodiments,the PUFA synthase comprises the amino acid sequence of SEQ ID NOs: 1, 2,or 3 or any combination thereof. In some embodiments, the nucleic acidsequence encoding the PUFA synthase comprises the nucleic acid sequenceof SEQ ID NOs: 6, 7 or 8 of any combination thereof.

In some embodiments, the PPTase comprises an amino acid sequence that is80% to 99% identical to SEQ ID NO:5 or comprises the amino acid sequenceof SEQ ID NO:5. In some embodiments, the nucleic acid sequence encodingthe PPTase is 80% to 99% identical to the nucleic acid sequence of SEQID NO:10 or comprises the nucleic acid sequence of SEQ ID NO:10.

In some embodiments, the nucleic acid sequences of (i) and (ii) arecontained in a single recombinant expression vector. In someembodiments, the nucleic acid sequences of (i) and (ii) are contained indifferent recombinant expression vectors. In some embodiments, thenucleic acid sequence(s) of (i) and/or (ii) are operably linked to aseed-specific promoter. In some embodiments, the nucleic acidsequence(s) of (i) and/or (ii) are operably linked to a promoterselected from PvDlec2, PvPhaseolin, LfKCS3, FAE 1, BoACP and BnaNapinC.In some embodiments, the nucleic acid sequence(s) of (i) and/or (ii) areoperably linked to a leaf-specific promoter. In some embodiments, thenucleic acid sequence(s) of (i) and/or (ii) are operably linked to aubiquitin or CsVMV promoter.

In some embodiments, the genetically modified plant, descendant, seed,cell, tissue, or part thereof further comprises (iii) a nucleic acidsequence encoding an acyl-CoA synthetase (ACoAS) that catalyzes theconversion of long chain PUFA free fatty acids (PFFA) to acyl-CoA. Insome embodiments, the ACoAS comprises an amino acid sequence that is 80%to 99% identical to SEQ ID NO:4 or comprises the amino acid sequence ofSEQ ID NO:4. In some embodiments, the ACoAS comprises a nucleic acidsequence that is 80% to 99% identical to the nucleic acid sequence ofSEQ ID NO:9 or comprises the nucleic acid sequence of SEQ ID NO:9. Insome embodiments, the nucleic acid sequence encoding the ACoAS comprisesthe nucleic acid sequence of SEQ ID NO:34. In some embodiments, thenucleic acid sequences of (i), (ii) and/or (iii) are contained in asingle recombinant expression vector. In some embodiments, the nucleicacid sequences of (i), (ii) and (iii) are contained in differentrecombinant expression vectors. In some embodiments, the nucleic acidsequences of (i) and (ii) are contained in a single recombinantexpression vector and the nucleic acid sequence of (iii) is contained ina different recombinant expression vector. In some embodiments, thenucleic acid sequences of (i) and (iii) are contained in a singlerecombinant expression vector and the nucleic acid sequence of (ii) iscontained in a different recombinant expression vector. In someembodiments, the nucleic acid sequences of (ii) and (iii) are containedin a single recombinant expression vector and the nucleic acid sequenceof (i) is contained in a different recombinant expression vector. Insome embodiments, the nucleic acid sequence(s) of (i), (ii) and/or (iii)are operably linked to a seed-specific promoter. In some embodiments,the nucleic acid sequence(s) of (i), (ii) and/or (iii) are operablylinked to a promoter selected from PvDlec2, LfKCS3, FAE 1, BoACP andBnaNapinC. In some embodiments, the nucleic acid sequence(s) of (i),(ii) and/or (iii) are operably linked to a leaf-specific promoter. Insome embodiments, the nucleic acid sequence(s) of (i), (ii) and/or (iii)are operably linked to a ubiquitin or CsVMV promoter.

In some embodiments, the genetically modified plant, descendant, cell,tissue, or part thereof further comprises a nucleic acid sequenceencoding an acetyl CoA carboxylase (ACCase) and/or a nucleic acidsequence encoding a type 2 diacylglycerol acyltransferase (DGAT2).

In some embodiments, the genetically modified plant, descendant, cell,tissue, seed or part thereof comprising at least one of pDAB7361,pDAB7362, pDAB7363, pDAB7368, pDAB7369, pDAB7370, pDAB100518,pDAB101476, pDAB101477, pDAB9166, pDAB9167, pDAB7379, pDAB7380,pDAB9323, pDAB9330, pDAB9337, pDAB9338, pDAB9344, pDAB9396, pDAB101412,pDAB7733, pDAB7734, pDAB101493, pDAB109507, pDAB109508, pDAB109509,pDAB9151, pDAB108207, pDAB108208, pDAB108209, pDAB9159, pDAB9147,pDAB108224, and pDAB108225.

In some embodiments, a genetically modified plant, descendant, cell,tissue, seed, or part thereof or an oil (e.g., a seed oil) obtained fromthe genetically modified plant, descendant, seed, cell, tissue, or partthereof comprises detectable amounts of DHA (docosahexaenoic acid(C22:6, n-3)), DPA(n-6) (docosapentaenoic acid (C22:5, n-6)) and/or EPA(eicosapentaenoic acid (C20:5, n-3)). In some embodiments, thegenetically modified plant, descendant, cell, tissue, seed, or partthereof or an oil (e.g., a seed oil) obtained from the geneticallymodified plant, descendant, seed, cell, tissue, or part thereofcomprises 0.01% to 15% DHA by weight of total fatty acids, 0.05% to 10%DHA by weight of total fatty acids, or 0.05% to 5% DHA by weight oftotal fatty acids. In some embodiments, the genetically modified plant,descendant, cell, tissue, seed, or part thereof or an oil (e.g., a seedoil) obtained from the genetically modified plant, descendant, seed,cell, tissue, or part thereof comprises 0.01% to 10% EPA by weight oftotal fatty acids, 0.05% to 5% EPA by weight of total fatty acids, or0.05% to 1% EPA by weight of total fatty acids. In some embodiments, thegenetically modified plant, descendant, cell, tissue, seed, or partthereof or an oil (e.g., a seed oil) obtained from the geneticallymodified plant, descendant, seed, cell, tissue, or part thereofcomprises 0.01% to 10% DPA(n-6) by weight of total fatty acids, 0.01% to5% DPA(n-6) by weight of total fatty acids, or 0.01% to 1% DPA(n-6) byweight of total fatty acids. In some embodiments, the geneticallymodified plant, descendant, cell, tissue, seed, or part thereof or anoil (e.g., a seed oil) obtained from the genetically modified plant,descendant, seed, cell, tissue, or part thereof comprises a ratio ofEPA:DHA of 1:1 to 1:10 or 1:1 to 1:3 by weight of total fatty acids. Insome embodiments, the genetically modified plant, descendant, cell,tissue, seed, or part thereof or an oil (e.g., a seed oil) obtained fromthe genetically modified plant, descendant, seed, cell, tissue, or partthereof comprises a ratio of DPA(n-6):DHA of 1:1 to 1:10 or 1:1 to 1:3by weight of total fatty acids. In some embodiments, the oil (e.g., aseed oil) obtained from a genetically modified plant, descendant, cell,tissue, seed, or part thereof comprises 70% to 99% triglycerides byweight of the oil.

In some embodiments, the detectable amounts of DHA, DPA(n-6) and/or EPAare also found in grain and/or meal obtained from the geneticallymodified plant, descendant, tissue, seed, or part thereof.

The present invention is directed to an oil (e.g., a seed oil) or a seedobtained from a genetically modified plant (e.g., soybean), descendant,cell, tissue, or part thereof described herein. The present invention isdirected to a food product comprising an oil (e.g., a seed oil) obtainedfrom a genetically modified plant (e.g., soybean), descendant, cell,tissue, or part thereof described herein. The present invention is alsodirected to a functional food comprising an oil (e.g., a seed oil) or aseed obtained from a genetically modified plant (e.g., soybean),descendant, cell, tissue, or part thereof described herein. The presentinvention is directed to a pharmaceutical product comprising an oil(e.g., a seed oil) or a seed obtained from a genetically modified plant(e.g., soybean), descendant, cell, tissue, or part described herein.

The present invention is directed to a method to produce an oilcomprising at least one LC-PUFA, comprising recovering oil from agenetically modified plant (e.g., soybean), descendant, cell, tissue, orpart thereof described herein or from a seed of a genetically modifiedplant (e.g., soybean), descendant, cell, tissue, or part thereofdescribed herein. The present invention is also directed to a method toproduce an oil comprising at least one LC-PUFA, comprising growing agenetically modified plant (e.g., soybean), descendant, cell, tissue, orpart thereof described herein. The present invention is also directed toa method to produce at least one LC-PUFA in a seed oil, comprisingrecovering oil from a seed of a genetically modified plant (e.g.,soybean), descendant, cell, tissue, or part thereof described herein.

The present invention is directed to a method to produce at least onePUFA in a seed oil, comprising growing a genetically modified plant(e.g., soybean), descendant, cell, tissue, or part thereof describedherein. The present invention is also directed to a method to provide asupplement or therapeutic product containing at least one PUFA to anindividual, comprising providing to the individual a geneticallymodified plant (e.g., soybean), descendant, cell, tissue, or partthereof of described herein, an oil described herein, a seed describedherein, a food product described herein, a functional food describedherein, or a pharmaceutical product described herein. In someembodiments, a PUFA contained in such embodiments is DHA, DPA(n-6)and/or EPA.

The present invention is directed to a method to produce a geneticallymodified plant (e.g., soybean), descendant, cell, tissue, or partthereof described herein, comprising transforming a plant or plant cellwith (i) a nucleic acid sequence encoding a PUFA synthase (e.g., analgal PUFA synthase) that produces at least one polyunsaturated fattyacid (PUFA); and (ii) a nucleic acid sequence encoding aphosphopantetheinyl transferase (PPTase) that transfers aphosphopantetheinyl cofactor to an PUFA synthase (e.g., an algal PUFAsynthase) ACP domain. In some embodiments, the method further comprisestransforming the plant or plant cell with (iii) a nucleic acid sequenceencoding an acyl-CoA synthetase (ACoAS) that catalyzes the conversion oflong chain PUFA free fatty acids (FFA) to acyl-CoA.

BRIEF DESCRIPTION OF DRAWINGS

The various embodiments of the invention can be more fully understoodfrom the following detailed description, the figures, and theaccompanying sequence descriptions, which form a part of thisapplication. These sequences include, in order: Repeat 1 (SEQ ID NO:65),Repeat 3 (SEQ ID NO: 66), Repeat 4 (SEQ ID NO:67), Repeat 8 (SEQ ID NO:68), Repeat 5 (SEQ ID NO:69), Repeat 9 (SEQ ID NO:70), Repeat 2 (SEQ IDNO:71), Repeat 6 (SEQ ID NO:72), and Repeat 7 (SEQ ID NO: 73).

FIG. 1 depicts the Clustal W (alignments in Vector NTI) of theredesigned DNA sequences encoding each of the 9 repeat domains of PUFAOrfA.

FIG. 2 is a plasmid map of pDAB7362.

FIG. 3 is a plasmid map of pDAB7361.

FIG. 4 is a plasmid map of pDAB7363.

FIG. 5 is a plasmid map of pDAB7365.

FIG. 6 is a plasmid map of pDAB7368.

FIG. 7 is a plasmid map of pDAB7369.

FIG. 8 is a plasmid map of pDAB7370.

FIG. 9 is a plasmid map of pDAB100518.

FIG. 10 is a plasmid map of pDAB101476.

FIG. 11 is a plasmid map of pDAB101477.

FIG. 12 shows the DHA and LC-PUFA content of single T2 soybean seedsfrom T1 plants derived from two soybean events transformed withpDAB7362.

FIG. 13 shows Western blot detection of PUFA synthase OrfA, PUFAsynthase OrfB, and PUFA synthase chimeric OrfC in T2 soybean seedprotein extracts.

FIG. 14 is a plasmid map of pDAB9166.

FIG. 15 is a plasmid map of pDAB9167.

FIG. 16 is a plasmid map of pDAB7379.

FIG. 17 is a plasmid map of pDAB7380.

FIG. 18 is a plasmid map of pDAB9323.

FIG. 19 is a plasmid map of pDAB9330.

FIG. 20 is a plasmid map of pDAB9337.

FIG. 21 is a plasmid map of pDAB9338.

FIG. 22 is a plasmid map of pDAB9344.

FIG. 23 is a plasmid map of pDAB9396.

FIG. 24 is a plasmid map of pDAB101412.

FIG. 25 is a plasmid map of pDAB7733.

FIG. 26 is a plasmid map of pDAB7734.

FIG. 27 is a plasmid map of pDAB101493.

FIG. 28 is a plasmid map of pDAB109507.

FIG. 29 is a plasmid map of pDAB109508.

FIG. 30 is a plasmid map of pDAB109509.

FIG. 31 is a plasmid map of pDAB9151.

FIG. 32 is a plasmid map of pDAB108207.

FIG. 33 is a plasmid map of pDAB108208.

FIG. 34 is a plasmid map of pDAB108209.

FIG. 35 is a plasmid map of pDAB9159.

FIG. 36 is a plasmid map of pDAB9147.

FIG. 37 is a plasmid map of pDAB108224.

FIG. 38 is a plasmid map of pDAB108225.

FIG. 39 shows DHA and LC-PUFA content of T2 seed from individualtransgenic Arabidopsis events transformed with pDAB101493, pDAB7362,pDAB7369, pDAB101412 or pDAB7380.

DETAILED DESCRIPTION OF THE INVENTION

The term “polyunsaturated fatty acid” or “PUFA” as used herein refers tofatty acids with a carbon chain length of at least 16 carbons, at least18 carbons, at least 20 carbons, or 22 or more carbons, with at least 3or more double bonds, 4 or more double bonds, 5 or more double bonds, or6 or more double bonds, wherein all double bonds are in the cisconfiguration.

The term “long chain polyunsaturated fatty acids” or “LC-PUFAs” as usedherein refers to fatty acids of 20 and more carbon chain length,containing 3 or more double bonds, or 22 or more carbons, with at least3 or more double bonds, 4 or more double bonds, 5 or more double bonds,or 6 or more double bonds. LC-PUFAs of the omega-6 series include, butare not limited to, di-homo-gamma-linolenic acid (C20:3n-6), arachidonicacid (C20:4n-6), adrenic acid (also called docosatetraenoic acid or DTA)(C22:4n-6), and docosapentaenoic acid (C22:5n-6). LC-PUFAs of theomega-3 series include, but are not limited to, eicosatrienoic acid(C20:3n-3), eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid(C20:5n-3), docosapentaenoic acid (C22:5n-3), and docosahexaenoic acid(C22:6n-3). LC-PUFAs also include fatty acids with greater than 22carbons and 4 or more double bonds including but not limited toC28:8(n-3).

The term “PUFA synthase” as used herein refers to an enzyme thatproduces polyunsaturated fatty acids (PUFAs) and particularly, longchain PUFAs (LC-PUFAs) as well as any domain of such an enzyme in acomplex. The term PUFA synthase includes, but is not limited to, PUFAPKS systems or PKS-like systems for the production of PUFAs. Somespecific PUFA synthases are designated herein by an additional notation,e.g., “SzPUFA” synthase or “hSzThPUFA” synthase, as defined in theapplication. The term “PUFA synthase system” includes a PUFA synthaseand any accessory enzymes that can affect the function of the PUFAsynthase when expressed in a heterologous organism (e.g., a PPTase orACS).

The terms “phosphopantetheinyl transferase” and “PPTase” as used hereinrefer to an enzyme that activates a PUFA synthase by transferring acofactor (e.g., 4-phosphopantetheine) from coenzyme A (CoA) to one ormore ACP domains present in the PUFA synthase. One example of a PPTasethat can activate one or more ACP domains of a PUFA synthase describedherein is the Het I protein of Nostoc sp. PCC 7120 (formerly calledAnabaena sp. PCC 7120), designated herein as “NoHetI.”

The terms “acyl-CoA synthetase,” “ACoAS” and “ACS” as used herein referto an enzyme that catalyzes the conversion of long chain polyunsaturatedfree fatty acids (FFA) to acyl-CoA. Some specific acyl-CoA synthetasesare designated herein by an additional notation, e.g., “SzACS-2,” asdefined in the application.

The term “plant” as used herein includes any descendant, cell, tissue,seed, seed oil, or part thereof.

“Nutraceutical” means a product isolated, purified, concentrated, orproduced from plants that provides a physiological benefit or providesprotection against disease, including processed foods supplemented withsuch products, along with foods produced from crops that have beengenetically engineered to contain enhanced levels of suchphysiologically-active components.

“Functional food” means a food that (a) is similar in appearance to orcan be a conventional food that is consumed as part of a usual diet and(b) has enhanced nutritional value and/or specific dietary benefitsbased on a modification in the proportion of components that typicallyexist in the unmodified food.

The terms “polynucleotide” and “nucleic acid” are intended to encompassa singular nucleic acid as well as plural nucleic acids, a nucleic acidmolecule or fragment, variant, or derivative thereof, or construct,e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide ornucleic acid can contain the nucleotide sequence of the full-length cDNAsequence, or a fragment thereof, including the untranslated 5′ and 3′sequences and the coding sequences. A polynucleotide or nucleic acid canbe composed of any polyribonucleotide or polydeoxyribonucleotide, whichcan be unmodified RNA or DNA or modified RNA or DNA. For example, apolynucleotide or nucleic acid can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis a mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that can be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions.These terms also embrace chemically, enzymatically, or metabolicallymodified forms of a polynucleotide or nucleic acid.

A polynucleotide or nucleic acid sequence can be referred to as“isolated” in which it has been removed from its native environment. Forexample, a heterologous polynucleotide or nucleic acid encoding apolypeptide or polypeptide fragment having dihydroxy-acid dehydrataseactivity contained in a vector is considered isolated for the purposesof the present invention. Further examples of an isolated polynucleotideor nucleic acid include recombinant polynucleotides maintained inheterologous host cells or a purified (partially or substantially)polynucleotide or nucleic acid in solution. An isolated polynucleotideor nucleic acid according to the present invention further includes suchmolecules produced synthetically. An isolated polynucleotide or nucleicacid in the form of a polymer of DNA can be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

The term “gene” refers to a nucleic acid or fragment thereof that iscapable of being expressed as a specific protein, optionally includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence.

As used herein, the term “coding region” refers to a DNA sequence thatcodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence and that influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences can include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site, and stem-loop structure.

As used herein, the terms “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides” and fragmentsthereof, and refers to a molecule composed of monomers (amino acids)linearly linked by amide bonds (also known as peptide bonds). The term“polypeptide” refers to any chain or chains of two or more amino acids,and does not refer to a specific length of the product. Thus, peptides,dipeptides, tripeptides, oligopeptides, protein, amino acid chain, orany other term used to refer to a chain or chains of two or more aminoacids, are included within the definition of “polypeptide,” and the term“polypeptide” can be used instead of, or interchangeably with any ofthese terms. A polypeptide can be derived from a natural biologicalsource or produced by recombinant technology, but is not necessarilytranslated from a designated nucleic acid sequence. It can be generatedin any manner, including by chemical synthesis.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposes of the invention, as are native orrecombinant polypeptides that have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, “native” refers to the form of a polynucleotide, gene orpolypeptide as found in nature with its own regulatory sequences, ifpresent.

As used herein, “endogenous” refers to the native form of apolynucleotide, gene or polypeptide in its natural location in theorganism or in the genome of an organism. “Endogenous polynucleotide”includes a native polynucleotide in its natural location in the genomeof an organism. “Endogenous gene” includes a native gene in its naturallocation in the genome of an organism. “Endogenous polypeptide” includesa native polypeptide in its natural location in the organism.

As used herein, “heterologous” refers to a polynucleotide, gene orpolypeptide not normally found in the host organism but that isintroduced into the host organism. “Heterologous polynucleotide”includes a native coding region, or portion thereof, that isreintroduced into the source organism in a form that is different fromthe corresponding native polynucleotide. “Heterologous gene” includes anative coding region, or portion thereof, that is reintroduced into thesource organism in a form that is different from the correspondingnative gene. For example, a heterologous gene can include a nativecoding region that is a portion of a chimeric gene including non-nativeregulatory regions that is reintroduced into the native host.“Heterologous polypeptide” includes a native polypeptide that isreintroduced into the source organism in a form that is different fromthe corresponding native polypeptide.

As used herein, the term “modification” refers to a change in apolynucleotide disclosed herein that results in reduced, substantiallyeliminated or eliminated activity of a polypeptide encoded by thepolynucleotide, as well as a change in a polypeptide disclosed hereinthat results in reduced, substantially eliminated or eliminated activityof the polypeptide. Such changes can be made by methods well known inthe art, including, but not limited to, deleting, mutating (e.g.,spontaneous mutagenesis, random mutagenesis, mutagenesis caused bymutator genes, or transposon mutagenesis), substituting, inserting,down-regulating, altering the cellular location, altering the state ofthe polynucleotide or polypeptide (e.g., methylation, phosphorylation orubiquitination), removing a cofactor, introduction of an antisenseRNA/DNA, introduction of an interfering RNA/DNA, chemical modification,covalent modification, irradiation with UV or X-rays, homologousrecombination, mitotic recombination, promoter replacement methods,and/or combinations thereof. Guidance in determining which nucleotidesor amino acid residues can be modified, can be found by comparing thesequence of the particular polynucleotide or polypeptide with that ofhomologous polynucleotides or polypeptides, e.g., yeast or bacterial,and maximizing the number of modifications made in regions of highhomology (conserved regions) or consensus sequences.

The term “derivative” as used herein, refers to a modification of asequence disclosed in the present invention. Illustrative of suchmodifications would be the substitution, insertion, and/or deletion ofone or more bases relating to a nucleic acid sequence of a codingsequence disclosed herein that preserve, slightly alter, or increase thefunction of a coding sequence disclosed herein in oil seed crop species.Such derivatives can be readily determined by one skilled in the art,for example, using computer modeling techniques for predicting andoptimizing sequence structure. The term “derivative” thus also includesnucleic acid sequences having substantial sequence homology with thedisclosed coding sequences herein such that they are able to have thedisclosed functionalities for use in producing LC-PUFAs of the presentinvention.

As used herein, the term “variant” refers to a polypeptide differingfrom a specifically recited polypeptide of the invention by amino acidinsertions, deletions, mutations, and substitutions, created using,e.g., recombinant DNA techniques, such as mutagenesis. Guidance indetermining which amino acid residues can be replaced, added, or deletedwithout abolishing activities of interest, can be found by comparing thesequence of the particular polypeptide with that of homologouspolypeptides and minimizing the number of amino acid sequence changesmade in regions of high homology (conserved regions) or by replacingamino acids with consensus sequences.

Alternatively, recombinant polynucleotide variants encoding these sameor similar polypeptides can be synthesized or selected by making use ofthe “redundancy” in the genetic code. Various codon substitutions, suchas silent changes that produce various restriction sites, can beintroduced to optimize cloning into a plasmid or viral vector forexpression. Mutations in the polynucleotide sequence can be reflected inthe polypeptide or domains of other peptides added to the polypeptide tomodify the properties of any part of the polypeptide.

Amino acid “substitutions” can be the result of replacing one amino acidwith another amino acid having similar structural and/or chemicalproperties, i.e., conservative amino acid replacements, or they can bethe result of replacing one amino acid with an amino acid havingdifferent structural and/or chemical properties, i.e., non-conservativeamino acid replacements. “Conservative” amino acid substitutions can bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid. Alternatively, “non-conservative” amino acidsubstitutions can be made by selecting the differences in polarity,charge, solubility, hydrophobicity, hydrophilicity, or the amphipathicnature of any of these amino acids. “Insertions” or “deletions” can bewithin the range of variation as structurally or functionally toleratedby the recombinant proteins. The variation allowed can be experimentallydetermined by systematically making insertions, deletions, orsubstitutions of amino acids in a polypeptide molecule using recombinantDNA techniques and assaying the resulting recombinant variants foractivity.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters can be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters can direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths can have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (e.g., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression” as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression can also refer totranslation of mRNA into a polypeptide.

The term “overexpression” as used herein, refers to expression that ishigher than endogenous expression of the same or related gene. Aheterologous gene is overexpressed if its expression is higher than thatof a comparable endogenous gene.

As used herein, the term “transformation” refers to the transfer of anucleic acid or fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “plasmid” and “vector” as used herein refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements can be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction that iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell.

As used herein, the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withoutaffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts refers to thealteration of codons in the gene or coding regions of the nucleic acidmolecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, more than one, or a significant numberof codons with one or more codons that are more frequently used in thegenes of that organism.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” that shows which codons encodewhich amino acids are reproduced herein in the table below. As a result,many amino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

The Standard Genetic Code

T C A G T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC Phe (F)TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L) TCA Ser (S) TAA Stop TGA StopTTG Leu (L) TCG Ser (S) TAG Stop TGG Trp (W) C CTT Leu (L) CCT Pro (P)CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R)CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P)CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACT Thr (T) AAT Asn (N)AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I)ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACG Thr (T) AAG Lys (K)AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G)GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A)GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference, or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable and can be adapted in a number of ways. See Nakamura et al.,Nucl. Acids Res. 28:292 (2000). By utilizing this or similar tables, oneof ordinary skill in the art can apply the frequencies to any givenpolypeptide sequence, and produce a nucleic acid fragment of acodon-optimized coding region that encodes the polypeptide, but usescodons optimal for a given species. The present invention pertains tocodon optimized forms of OrfA, OrfB, chimeric OrfC, PPTase and/or otheraccessory proteins of the invention, as described further herein.

The term “percent identity” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case can be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those disclosed in: 1) Computational MolecularBiology (A. M. Lesk, Ed.) Oxford University: NY (1988); 2) Biocomputing:Informatics and Genome Projects (D. W. Smith, Ed.) Academic: NY (1993);3) Computer Analysis of Sequence Data, Part I (A. M. Griffin and H. G.Griffin, Eds.) Humania: NJ (1994); 4) Sequence Analysis in MolecularBiology (G. von Heinje, Ed.) Academic (1987); and 5) Sequence AnalysisPrimer (M. Gribskov and J. Devereux, Eds.) Stockton: NY (1991).

Methods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations can be performed,for example, using the AlignX program of the Vector NTI® suite(Invitrogen, Carlsbad, Calif.) or MEGALIGN™ program of the LASERGENE®bioinformatics computing suite (DNASTAR, Inc., Madison, Wis.). Multiplealignment of the sequences is performed using the “Clustal method ofalignment,” which encompasses several varieties of the algorithmincluding the “Clustal V method of alignment” corresponding to thealignment method labeled Clustal V (disclosed by Higgins and Sharp,CABIOS. 5:151-153 (1989); D. G. Higgins, et al., Comput. Appl. Biosci.,8:189-191 (1992)) and found in the MEGALIGN™ program of the LASERGENE®bioinformatics computing suite (DNASTAR, Inc.). For multiple alignments,the default values correspond to GAP PENALTY=10 and GAP LENGTH PEN ALTY=10. Default parameters for pairwise alignments and calculation ofpercent identity of protein sequences using the Clustal method areKTUPLE-T, GAP PENALTY=3, WTNDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters are KTUPLE-2, GAP PENALTY-5, WINDOW=4 andDIAGONALS SAVED=4. After alignment of the sequences using the Clustal Vprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” table in the same program. Additionally the“Clustal W method of alignment” is available and corresponds to thealignment method labeled Clustal W (described by Higgins and Sharp,CABIOS. 5:151-153 (1989); D.G. Higgins, et al., Comput. Appl. Biosci.8:189-191 (1992)) and found in the MEGALIGN™ v6.1 program of theLASERGENE® bioinformatics computing suite (DNASTAR, Inc.). Defaultparameters for multiple alignment (GAP PENALTY™ 10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” can be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR® (DNASTAR, Inc. Madison, Wis.); 4.)SEQUENCHER® (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) theFASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 11 1-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described, e.g., by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); and by Silhavyet al., Experiments with Gene Fusions, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1984); and by Ausubel et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987 to present).

The genetic manipulations of a recombinant hosts disclosed herein can beperformed using standard genetic techniques and screening and can bemade in any host cell that is suitable to genetic manipulation. In someembodiments, a recombinant host can be, but is not limited to, anyhigher plant, including both dicotyledonous and monocotyledonous plants,and consumable plants, including crop plants and plants used for theiroils. Thus, any plant species or plant cell can be selected as describedfurther below.

The oils of the present invention can also be used in non-culinary ordietary processes and compositions. Some of these uses can beindustrial, cosmetic or medical. Oils of the present invention can alsobe used in any application for which the oils of the present inventionare suited. In general, the oils of the present invention can be used toreplace, e.g., mineral oils, esters, fatty acids, or animal fats in avariety of applications, such as lubricants, lubricant additives, metalworking fluids, hydraulic fluids and fire resistant hydraulic fluids.The oils of the present invention can also be used as materials in aprocess of producing modified oils. Examples of techniques for modifyingoils of the present invention include fractionation, hydrogenation,alteration of the oil's oleic acid or linolenic acid content, and othermodification techniques known to those of skill in the art.

Examples of cosmetic uses for oils of the present invention include useas an emollient in a cosmetic composition; as a petroleum jellyreplacement; as comprising part of a soap, or as a material in a processfor producing soap; as comprising part of an oral treatment solution; ascomprising part of an ageing treatment composition; and as comprisingpart of a skin or hair aerosol foam preparation.

Additionally, the oils of the present invention can be used in medicalapplications. For example, oils of the present invention can be used ina protective barrier against infection, and oils high in omega-9 fattyacids can be used to enhance transplant graft survival (U.S. Pat. No.6,210,700).

It should be understood that the foregoing are non-limiting examples ofnon-culinary uses for which the oils of the present invention aresuited. As previously stated, oils and modified oils of the presentinvention can be used to replace, e.g., mineral oils, esters, fattyacids, or animal fats in all applications known to those of skill in theart.

PUFA Synthase

The “standard” or “classical” pathway for synthesis of long chain PUFAs(LC-PUFAs) in eukaryotic organisms involves the elongation anddesaturation of medium chain-length saturated or mono-unsaturated fattyacids and has been described. The pathway for synthesis of long chainPUFAs via a PUFA synthase has also been described and is very differentfrom the “standard” pathway. Specifically, PUFA synthases utilizemalonyl-CoA as a carbon source and produce the final PUFA withoutreleasing intermediates in any significant amount. Also, with PUFAsynthases, the appropriate cis double bonds are added during thesynthesis using a mechanism that does not require oxygen. In someembodiments, NADPH is used as a reductant during the synthesis cycles.

The present invention relates to host organisms (e.g., plants such assoybean) that have been genetically modified to express a PUFA synthase(either endogenously or by genetic manipulation). In some embodiments,an organism that has been genetically modified to express a PUFAsynthase, wherein the organism does not naturally (endogenously, withoutgenetic modification) express such an enzyme, or at least thatparticular PUFA synthase or portion thereof with which the organism isbeing genetically modified, can be referred to herein as a“heterologous” host organism with regard to the modification of theorganism with the PUFA synthase or with another protein that is notendogenously expressed by the organism. The genetic modifications of thepresent invention can be used to improve PUFA production in a hostorganism that endogenously expresses a PUFA synthase, where the organismis not further modified with a different PUFA synthase or a portionthereof.

A PUFA synthase according to the present invention can comprise severalmultifunctional proteins (and can include single function proteins,particularly for PUFA synthase from marine bacteria) that can acttogether to conduct both iterative processing of the fatty acid chain aswell as non-iterative processing, including trans-cis isomerization andenoyl reduction reactions in selected cycles. These proteins can also bereferred to herein as the core PUFA synthase enzyme complex or the corePUFA synthase. The general functions of the domains and motifs containedwithin these proteins are individually known in the art and have beendescribed in detail with regard to various PUFA synthases from marinebacteria and eukaryotic organisms (see, e.g., U.S. Pat. No. 6,140,486;U.S. Pat. No. 6,566,583; Metz et al., Science 293:290-293 (2001); U.S.Appl. Pub. No. 2002/0194641, now U.S. Pat. No. 7,247,461, issued July24, 2007; U.S. Appl. Pub. No. 2004/0235127, now U.S. Pat. No. 7,211,418,issued May 1, 2007; U.S. Appl. Pub. No. 2005/0100995, now U.S. Pat. No.7,217,856, issued May 15, 2007; and WO 2006/135866). The domains can befound as a single protein (e.g., the domain and protein are synonymous)or as one of two or more (multiple) domains in a single protein, asmentioned above. The domain architecture of various PUFA synthases frommarine bacteria and members of Thraustochytrium, and the structural andfunctional characteristics of genes and proteins comprising such PUFAsynthases, have been described (see, e.g., U.S. Pat. No. 6,140,486; U.S.Pat. No. 6,566,583; Metz et al., Science 293:290-293 (2001); U.S. Appl.Pub. No. 2002/0194641, now U.S. Pat. No. 7,247,461, issued Jul. 24,2007; U.S. Appl. Pub. No. 2004/0235127, now U.S. Pat. No. 7,211,418,issued May 1, 2007; U.S. Appl. Pub. No. 2005/0100995, now U.S. Pat. No.7,217,856, issued May 15, 2007; and WO 2006/135866).

Numerous examples of polynucleotides, genes and polypeptides having PUFAsynthase activity are known in the art and can be used in a geneticallymodified host disclosed herein. PUFA synthase proteins or domains thatare useful in the present invention can include both bacterial andnon-bacterial PUFA synthases. A non-bacterial PUFA synthase is a systemthat is from or derived from an organism that is not a bacterium, suchas a eukaryote. Bacterial PUFA synthases are described, for example, inU.S. Appl. Pub. No. 2008/0050505, now U.S. Pat. No. 7,868,228, issuedJan. 11, 2011. Genetically modified plants of the invention can beproduced that incorporate non-bacterial PUFA synthase functional domainswith bacterial PUFA synthase functional domains, as well as PUFAsynthase functional domains or proteins from other PKS systems (Type Iiterative or modular, Type II, or Type III) or FAS systems.

In some embodiments, a PUFA synthase of the present invention comprisesat least the following biologically active domains that are typicallycontained on three, four, or more proteins: (a) at least one enoyl-ACPreductase (ER) domain; (b) multiple acyl carrier protein (ACP) domain(s)(e.g., at least from one to four, or at least five ACP domains, and insome embodiments up to six, seven, eight, nine, ten, or more than tenACP domains); (c) at least two β-ketoacyl-ACP synthase (KS) domains; (d)at least one acyltransferase (AT) domain; (e) at least oneβ-ketoacyl-ACP reductase (KR) domain; (f) at least two FabA-likeβ-hydroxyacyl-ACP dehydrase (DH) domains; (g) at least one chain lengthfactor (CLF) domain; and/or (h) at least one malonyl-CoA:ACPacyltransferase (MAT) domain. In some embodiments, a PUFA synthaseaccording to the present invention also comprises at least one regioncontaining a dehydratase (DH) conserved active site motif

In some embodiments, a PUFA synthase comprises at least the followingbiologically active domains: (a) at least one enoyl-ACP reductase (ER)domain; (b) at least five acyl carrier protein (ACP) domains; (c) atleast two β-ketoacyl-ACP synthase (KS) domains; (d) at least oneacyltransferase (AT) domain; (e) at least one β-ketoacyl-ACP reductase(KR) domain; (f) at least two FabA-like β-hydroxyacyl-ACP dehydrase (DH)domains; (g) at least one chain length factor (CLF) domain; and (h) atleast one malonyl-CoA:ACP acyltransferase (MAT) domain. In someembodiments, a PUFA synthase according to the present invention alsocomprises at least one region or domain containing a dehydratase (DH)conserved active site motif that is not a part of a FabA-like DH domain.The structural and functional characteristics of each of these domainsare described in detail in U.S. Appl. Pub. No. 2002/0194641, now U.S.Pat. No. 7,247,461, issued Jul. 24, 2007; U.S. Appl. Pub. No.2004/0235127, now U.S. Pat. No. 7,211,418, issued May 1, 2007; U.S.Appl. Pub. No. 2005/0100995, now U.S. Pat. No. 7,217,856, issued May 15,2007; U.S. Appl. Pub. No. 2007/0245431; and WO 2006/135866.

There are three open reading frames that form the core SchizochytriumPUFA synthase and that have been described previously, e.g., in U.S.Appl. Pub. No. 2007/0245431. The domain structure of each open readingframe is as follows.

Schizochytrium Open Reading Frame A (OrfA or Pfa1): OrfA is a 8730nucleotide sequence (not including the stop codon) that encodes a 2910amino acid sequence. Within OrfA, there are twelve domains: (a) oneβ-keto acyl-ACP synthase (KS) domain; (b) one malonyl-CoA:ACPacyltransferase (MAT) domain; (c) nine acyl carrier protein (ACP)domains; and (d) one ketoreductase (KR) domain. Genomic DNA clones(plasmids) encoding OrfA from both Schizochytrium sp. ATCC 20888 and adaughter strain of ATCC 20888, denoted Schizochytrium sp., strain N230D,have been isolated and sequenced.

Genomic clone pJK1126 (denoted pJK1126 OrfA genomic clone, in the formof an E. coli plasmid vector containing “OrfA” gene from SchizochytriumATCC 20888) was deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 201 10-2209 USA onJun. 8, 2006, and assigned ATCC Accession No. PTA-7648.

Genomic clone pJK306 (denoted pJK306 OrfA genomic clone, in the form ofan E. coli plasmid containing 5′ portion of OrfA gene fromSchizochytrium sp. N230D (2.2 kB overlap with pJK320)) was depositedwith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assignedATCC Accession No. PTA-7641.

Genomic clone pJK320 (denoted pJK320 OrfA genomic clone, in the form ofan E. coli plasmid containing 3′ portion of OrfA gene fromSchizochytrium sp. N230D (2.2 kB overlap with pJK306)) was depositedwith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 201 10-2209 USA on Jun. 8, 2006, and assignedATCC Accession No. PTA-7644.

Schizochytrium Open Reading Frame B (OrfB or Pfa2): OrfB is a 6177nucleotide sequence (not including the stop codon) that encodes a 2059amino acid sequence. Within OrfB, there are four domains: (a) one-ketoacyl-ACP synthase (KS) domain; (b) one chain length factor (CLF) domain;(c) one acyl transferase (AT) domain; and, (d) one enoyl ACP-reductase(ER) domain. Genomic DNA clones (plasmids) encoding OrfB from bothSchizochytrium sp. ATCC 20888 and a daughter strain of ATCC 20888,denoted Schizochytrium sp., strain N230D, have been isolated andsequenced.

Genomic clone pJK1129 (denoted pJK1129 OrfB genomic clone, in the formof an E. coli plasmid vector containing “OrfB” gene from SchizochytriumATCC 20888) was deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun.8, 2006, and assigned ATCC Accession No. PTA-7649.

Genomic clone pJK324 (denoted pJK324 OrfB genomic clone, in the form ofan E. coli plasmid containing the OrfB gene sequence from Schizochytriumsp. N230D) was deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun.8, 2006, and assigned ATCC Accession No. PTA-7643.

Schizochytrium Open Reading Frame C (OrfC or Pfa3): OrfC is a 4506nucleotide sequence (not including the stop codon) that encodes a 1502amino acid sequence. Within OrfC, there are three domains: (a) twoFabA-like-hydroxy acyl-ACP dehydrase (DH) domains; and (b) one enoylACP-reductase (ER) domain. Genomic DNA clones (plasmids) encoding OrfCfrom both Schizochytrium sp. ATCC 20888 and a daughter strain of ATCC20888, denoted Schizochytrium sp., strain N230D, have been isolated andsequenced.

Genomic clone pJK1131 (denoted pJK1131 OrfC genomic clone, in the formof an E. coli plasmid vector containing “OrfC” gene from SchizochytriumATCC 20888) was deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 201 10-2209 USA onJun. 8, 2006, and assigned ATCC Accession No. PTA-7650.

Genomic clone pBR002 (denoted pBR002 OrfC genomic clone, in the form ofan E. coli plasmid vector containing the OrfC gene sequence fromSchizochytrium sp. N230D) was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209USA on Jun. 8, 2006, and assigned ATCC Accession No. PTA-7642.

In addition, there are three open reading frames that form the coreThraustochytrium PUFA synthase that have been described previously. Thedomain structure of each open reading frame is as follows.

Thraustochytrium 23 Open Reading Frame A (OrfA): OrfA is a 8433nucleotide sequence (not including the stop codon) that encodes a 281 1amino acid sequence. The following domains are present in Th. 23B OrfA:(a) one β-ketoacyl-ACP synthase (KS) domain; (b) one malonyl-CoA:ACPacyltransferase (MAT) domain; (c) eight acyl earner protein (ACP)domains; and (d) one β-ketoacyl-ACP reductase (R) domain.

Genomic clone Th23BOrfA_pBR812.1 (denoted Th23BOrfA_pBR812.1 genomicclone, in the form of an E. coli plasmid vector containing the OrfA genesequence from Thraustochytrium 23 B) was deposited with the AmericanType Culture Collection (ATCC), University Boulevard, Manassas, Va. 20110-2209 USA on Mar. 1, 2007, and assigned ATCC Accession No. PTA-8232.Genomic clone Th23BOrfA_pBR811 (denoted Th23BOrfA_pBR811 genomic clone,in the form of an E. coli plasmid vector containing the OrfA genesequence from Thraustochytrium 23B) was deposited with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Mar. 1, 2007, and assigned ATCC Accession No. PTA-8231.

Thraustochytrium 23B Open Reading Frame B (OrfB): OrfB is a 5805nucleotide sequence (not including the stop codon) that encodes a 1935amino acid sequence. The following domains are present in Th. 23B OrfB:(a) one β-ketoacyl-ACP synthase (KS) domain; (b) one chain length factor(CLF) domain; (c) one acyltransferase (AT) domain; and, (d) oneenoyl-ACP reductase (ER) domain. Genomic clone Th23BOrfBpBR800 (denotedTh23BOrfBpBR800 genomic clone, in the form of an E. coli plasmid vectorcontaining the OrfB gene sequence from Thraustochytrium 23 B) wasdeposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 USA on Mar. 1, 2007, andassigned ATCC Accession No. PTA-8227.

Thraustochytrium 23B Open Reading Frame C (OrfC): OrfC is a 4410nucleotide sequence (not including the stop codon) that encodes a 1470amino acid sequence. The following domains are present in Th. 23B OrfC:(a) two FabA-like β-hydroxyacyl-ACP dehydrase (DH) domains, both withhomology to the FabA protein (an enzyme that catalyzes the synthesis oftrans-2-decenoyl-ACP and the reversible isomerization of this product tocis-3-decenoyl-ACP); and (b) one enoyl-ACP reductase (ER) domain withhigh homology to the ER domain of Schizochytrium OrfB. Genomic cloneTh23BOrfC_pBR709A (denoted Th23BOrfC_pBR709A genomic clone, in the formof an E. coli plasmid vector containing the OrfC gene sequence fromThraustochytrium 23B) was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 201 10-2209USA on Mar. 1, 2007, and assigned ATCC Accession No. PTA-8228.

Chimeric or hybrid PUFA synthase: In some embodiments, the PUFA synthasecomprises domains selected from any of those described herein, whereinthe domains are combined (e.g., mixed and matched) to form a completePUFA synthase meeting the minimum requirements described herein. In someembodiments, the genetically modified organism of the invention can befurther modified with at least one domain or biologically activefragment thereof of another PUFA synthase. In some embodiments, any ofthe domains of a PUFA synthase can be modified from their naturalstructure to modify or enhance the function of that domain in the PUFAsynthase system (e.g., to modify the PUFA types or ratios thereofproduced by the system). Such mixing of domains to produce chimeric PUFAsynthase is described in the patents and publications referenced herein.

In some embodiments, the PUFA synthase comprises a Schizochytrium PUFAsynthase wherein OrfC from the Schizochytrium PUFA synthase is replacedwith OrfC from Thraustochytrium 23B. In some embodiments, such achimeric OrfC from Thraustochytrium 23B is encoded by a nucleic acidsequence that is optimized for Schizochytrium codon usage. As anon-limiting example of such a chimeric OrfC, plasmid pThOrfC-synPS(denoted pThOrfC-synPS, in the form of an E. coli plasmid vectorcontaining a “perfect stitch” synthetic Thraustochytrium 23 B PUFA PKSOrfC codon optimized for expression in Schizochytrium or otherheterologous hosts) was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 201 10-2209USA on Mar. 1, 2007, and assigned ATCC Accession No. PTA-8229 (see alsoU.S. Appl. Pub. No. 2008/0022422, now U.S. Pat. No. 8,003,772, issuedAug. 23, 2011).

Other examples of PUFA synthase genes and polypeptides that can be usedin a genetically modified organism of the invention include, but are notlimited to, the following codon-optimized sequences generated by themethods described further herein: SEQ ID NO:1 (SzPUFA OrfA v3 protein);SEQ ID NO:2 (SzPUFA OrfB v3 protein); SEQ ID NO:3 (hSzThPUFA OrfC v3protein); SEQ ID NO:6 (SzPUFA OrfA gene); SEQ ID NO:7 (SzPUFA OrfB v3gene); and SEQ ID NO:8 (hSzThPUFA OrfC v3 gene), as well as an activevariant, portion, fragment, or derivative of such sequences, whereinsuch a gene encodes, or such a polypeptide or protein has, PUFA synthaseactivity. The present invention includes an isolated polynucleotide orpolypeptide comprising or consisting of one or more of such sequences.

Other examples of PUFA synthase genes and polypeptides that can be usedin the invention include, but are not limited to, PUFA synthase genes orpolypeptides having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to any one of the PUFA synthase genes or polypeptidesdescribed herein. Useful ranges can be selected between any of thesevalues (for example, 80% to 100% identical, 85% to 100% identical, 90%to 100% identical, 95% to 100% identical, 80% to 99% identical, 85% to99% identical, 90% to 99% identical, or 95% to 99% identical). Stillother examples of PUFA synthase genes and polypeptides that can used ina genetically modified organism of the invention include, but are notlimited to an active variant, portion, fragment of derivative of any oneof the PUFA synthases or sequences described herein, wherein such a geneencodes, or such a polypeptide has, PUFA synthase activity.

In some embodiments, the PUFA synthase can be an algal PUFA synthase. Insome embodiments, the PUFA synthase can comprise an amino acid sequencethat is 80% to 100% identical, 85% to 100% identical, 90% to 100%identical, 95% to 100% identical, 80% to 99% identical, 85% to 99%identical, 90% to 99% identical, or 95% to 99% identical to the aminoacid sequence of SEQ ID NO:1. In some embodiments, the PUFA synthase cancomprise the amino acid sequence of SEQ ID NO:1. In some embodiments,the nucleic acid sequence encoding the PUFA synthase can comprise anucleic acid sequence 80% to 100% identical, 85% to 100% identical, 90%to 100% identical, 95% to 100% identical, 80% to 99% identical, 85% to99% identical, 90% to 99% identical, or 95% to 99% identical to thenucleic acid sequence of SEQ ID NO:6. In some embodiments, the nucleicacid sequence encoding the PUFA synthase can comprise the nucleic acidsequence of SEQ ID NO:6. In some embodiments, the PUFA synthase cancomprise an amino acid sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO:2. In someembodiments, the PUFA synthase can comprise the amino acid sequence ofSEQ ID NO:2. In some embodiments, the nucleic acid sequence encoding thePUFA synthase can comprise a nucleic acid sequence that is at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to the nucleic acid sequence of SEQID NO:7. in some embodiments, the nucleic acid sequence encoding thePUFA synthase can comprise the nucleic acid sequence of SEQ ID NO:7. Insome embodiments, the PUFA synthase can comprise an amino acid sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to the aminoacid sequence of SEQ ID NO:3. In some embodiments, the PUFA synthasecomprises the amino acid sequence of SEQ ID NO:3. In some embodiments,the nucleic acid sequence encoding the PUFA synthase comprises a nucleicacid sequence that is at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto the nucleic acid sequence of SEQ ID NO:8. In some embodiments, thenucleic acid sequence encoding the PUFA synthase comprises the nucleicacid sequence of SEQ ID NO:8.

In some embodiments, the PUFA synthase comprises the amino acid sequenceof SEQ ID NO:1, 2, or 3 or any combinations thereof. In someembodiments, the PUFA synthase comprises the nucleic acid sequence ofSEQ ID NO:6, 7, or 8 or any combinations thereof In some embodiments,the nucleic acid sequence encodes an amino acid sequence of SEQ ID NO:1,2, or 3, or any combinations or percent identities thereof describedherein.

In some embodiments, the sequences of other PUFA synthase genes and/orpolypeptides can be identified in the literature and in bioinformaticsdatabases well known to the skilled person using sequences disclosedherein and available in the art. For example, such sequences can beidentified through BLAST searching of publicly available databases withknown PUFA synthase gene or polypeptide sequences. In such a method,identities can be based on the Clustal W method of alignment using thedefault parameters of GAP PEN ALT Y=10, GAP LENGTH PENALTY-0.1, andGonnet 250 series of protein weight matrix.

Additionally, the PUFA synthase gene or polypeptide sequences disclosedherein or known the art can be used to identify other PUFA synthasehomologs in nature. For example, each of the PUFA synthase nucleic acidfragments disclosed herein can be used to isolate genes encodinghomologous proteins. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to (1) methodsof nucleic acid hybridization; (2) methods of DNA and RNA amplification,as exemplified by various uses of nucleic acid amplificationtechnologies (e.g., polymerase chain reaction (PCR), Mullis et al., U.S.Patent No. 4,683,202; ligase chain reaction (LCR), S. Tabor, et al.,Proc. Acad. Sci. USA 82:1074 (1985); or strand displacementamplification (SDA), Walker et al., Proc. Natl. Acad. Sci. U.S.A.,89:392 (1992)); and (3) methods of library construction and screening bycomplementation.

All of these methods can be readily practiced by one skilled in the artmaking use of the known or identified sequences encoding targetproteins. In some embodiments, DNA sequences surrounding a target PUFAsynthase coding sequence are also useful in some modification proceduresand can be readily found by one of skill in the art in publiclyavailable databases. Methods for creating genetic mutations are commonand well known in the art and can be applied to the exercise of creatingmutants.

Phosphopantethienyl Transferase

The phosphopantethienyl transferases (PPTases) are a family of enzymesthat have been well characterized in fatty acid synthesis, polyketidesynthesis, and non-ribosomal peptide synthesis. In particular, the ACPdomains present in the PUFA synthase enzymes require activation byattachment of a cofactor (4-phosphopantetheine) from coenzyme A to theacyl carrier protein (ACP). Attachment of this cofactor is carried outby PPTases. If the endogenous PPTases of the host organism are incapableof activating the PUFA synthase ACP domains, then it is necessary toprovide a PPTase that is capable of carrying out that function. Thesequences of many PPTases are known, and crystal structures have beendetermined (e.g., Reuter et al., EMBO J. 18:6823-31 (1999)) as well asmutational analysis of amino acid residues important for activity (Mofidet al., Biochemistry 43:4128-36 (2004)).

One example of a heterologous PPTase that has been demonstratedpreviously to recognize the OrfA ACP domains described herein assubstrates is the Het I protein of Nostoc sp. PCC 7120 (formerly calledAnabaena sp. PCC 7120). Het I is present in a cluster of genes in Nostocknown to be responsible for the synthesis of long chain hydroxy-fattyacids that are a component of a glyco-lipid layer present in heterocystsof that organism (Black and Wolk, J. Bacteriol. 775:2282-2292 (1994);Campbell et al., Arch. Microbiol. 7(57:251-258 (1997)). Het I is likelyto activate the ACP domains of a protein, Hgl E, present in thatcluster. The two ACP domains of Hgl E have a high degree of sequencehomology to the ACP domains found in Schizochytrium Orf A and other PUFAsynthases.

In some embodiments, a PUFA synthase can be considered to include atleast one 4′-phosphopantetheinyl transferase (PPTase) domain, or such adomain can be considered to be an accessory domain or protein to thePUFA synthase. Structural and functional characteristics of PPTases havebeen described in detail, for example, in U.S. Appl. Pub. No.2002/0194641, now U.S. Pat. No. 7,247,461, issued Jul. 24, 2007; U.S.Appl. Pub. No. 2004/0235127, now U.S. Pat. No. 7,211,418, issued May 1,2007; and U.S. Appl. Pub. No. 2005/0100995, now U.S. Pat. No. 7,217,856,issued May 15, 2007.

Numerous examples of genes and polypeptides having PPTase activity areknown in the art and could be used in a genetically modified organism ofthe invention if they are capable of activating the ACP domains of theparticular PUFA synthase being used. Examples of genes and polypeptidesthat can be used in a genetically modified organism of the invention caninclude, but are not limited to, the following codon-optimized sequencesdescribed further herein: SEQ ID NO:5 (NoHetI v3 protein) and SEQ IDNO:10 (NoHetI v3 gene).

Other examples of PPTase genes and polypeptides that can be used in agenetically modified organism of the invention include, but are notlimited to, PPTase genes or polypeptides having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to any one of the PPTases orsequences described herein. Useful ranges can be selected between any ofthese values (for example, 80% to 100% identical, 85% to 100% identical,90% to 100% identical, 95% to 100% identical, 80% to 99% identical, 85%to 99% identical, 90% to 99% identical, or 95% to 99% identical). Stillother examples of PPTase genes and polypeptides that can used in agenetically modified organism of the invention include, but are notlimited to an active variant, fragment, portion or derivative of any oneof the PPTase sequences described herein, wherein such a gene encodes,or such a polypeptide has, PPTase activity.

In some embodiments, the PPTase can be an algal PPTase. In someembodiments, the PPTase can comprise an amino acid sequence that is 80%to 100% identical, 85%» to 100% identical, 90% to 100% identical, 95% to100% identical, 80% to 99% identical, 85% to 99% identical, 90% to 99%identical, or 95% to 99% identical to the amino acid sequence of SEQ IDNO:5. In some embodiments, the PPTase can comprise the amino acidsequence of SEQ ID NO:5. In some embodiments, the nucleic acid sequenceencoding the PPTase can comprise a nucleic acid sequence 80% to 100%identical, 85% to 100% identical, 90% to 100% identical, 95% to 100%identical, 80% to 99% identical, 85% to 99% identical, 90% to 99%identical, or 95% to 99% identical to the nucleic acid sequence of SEQID NO:10. In some embodiments, the nucleic acid sequence encoding thePPTase can comprise the nucleic acid sequence of SEQ ID NO:10. In someembodiments, the nucleic acid sequence encodes an amino acid sequence ofSEQ ID NO:5 or any percent identities thereof described herein.

In some embodiments of the present invention, a PPTase can be providedfor production and/or accumulation of PPTase in a heterologous host.

In some embodiments, a gene and/or polypeptide encoding PPTase can beused to identify another PPTase gene and/or polypeptide sequences and/orcan be used to identify a PPTase homolog in other cells. Such PPTaseencoding sequences can be identified, for example, in the literatureand/or in bioinformatics databases well known to the skilled person. Forexample, the identification of a PPTase encoding sequence in anothercell type using bioinformatics can be accomplished through BLAST (asdisclosed above) searching of publicly available databases with a knownPPTase encoding DNA and polypeptide sequence, such as any of thoseprovided herein. Identities are based on the Clustal W method ofalignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix.

In some embodiments, the genetically modified plant (e.g., soybean),descendant, cell, tissue, or part thereof contains a PUFA synthase and aPPTase. In some embodiments, the genetically modified plant (e.g.,soybean), descendant, cell, tissue, or part thereof contains the nucleicacid sequences of (i) and (ii) in a single recombinant expressionvector. In some embodiments, the genetically modified plant (e.g.,soybean), descendant, cell, tissue, or part thereof contains the nucleicacid sequences of (i) and (ii) in different recombinant expressionvectors.

Acyl-CoA Synthetase

The present invention provides acyl-CoA synthetase (ACoAS) proteins thatcatalyze the conversion of long chain PUFA free fatty acids (FFA) toacyl-CoA. The endogenous producer of PUFAs by PUFA synthase,Schizochytrium, possesses one or more ACoASs that are capable ofconverting the free fatty acid products of its PUFA synthase intoacyl-CoA. Therefore, Schizochytrium, as well as other organisms thatendogenously contain a PUFA synthase (e.g., other Thraustochytrids) orother organisms that can convert PUFA FFAs into acyl-CoAs (such asThalassiosira pseudonana or Crypthecodinium cohnii), could representsources for genes encoding enzymes that are useful in permitting orincreasing the accumulation of the products of a PUFA synthase expressedin a heterologous host. Other ACoAS sequences have been described inU.S. Appl. Pub. No. 2007/0245431.

Numerous examples of genes and polypeptides having ACoAS activity areknown in the art and can be used in a genetically modified organism ofthe invention. Examples of genes and polypeptides that can be used in agenetically modified organism of the invention can include, but are notlimited to, the following codon-optimized sequences described furtherherein: SEQ ID NO:4 (SzACS-2 v3 protein) and SEQ ID NO:9 (hSzThACS-2 v3gene).

Other examples of ACoAS genes and polypeptides that can be used in agenetically modified organism of the invention include, but are notlimited to, ACoAS genes or polypeptides having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to any one of the ACoAS orsequences described herein. Useful ranges can be selected between any ofthese values (for example, 80% to 100% identical, 85% to 100% identical,90% to 100% identical, 95% to 100% identical, 80% to 99% identical, 85%to 99% identical, 90% to 99% identical, or 95% to 99% identical). Stillother examples of ACoAS genes and polypeptides that can used in agenetically modified organism of the invention include, but are notlimited to an active variant, fragment, portion, or derivative of anyone of the ACoAS sequences described herein, wherein such a geneencodes, or such a polypeptide has, ACoAS activity.

In some embodiments, the ACoAS can be an algal ACoAS. In someembodiments, the ACoAS can comprise an amino acid sequence that is 80%to 100% identical, 85% to 100% identical, 90% to 100% identical, 95% to100% identical, 80% to 99% identical, 85% to 99% identical, 90% to 99%identical, or 95% to 99% identical to the amino acid sequence of SEQ IDNO:4. In some embodiments, the ACoAS can comprise the amino acidsequence of SEQ ID NO:4. In some embodiments, the nucleic acid sequenceencoding the ACoAS can comprise a nucleic acid sequence 80% to 99%identical, 85% to 99% identical, 90% to 99% identical, 80% to 95%identical, or 85% to 95% identical to the nucleic acid sequence of SEQID NO:9. In some embodiments, the nucleic acid sequence encoding theACoAS can comprise the nucleic acid sequence of SEQ ID NO:9. In someembodiments, the nucleic acid sequence encoding the ACoAS comprises thenucleic acid sequence of SEQ ID NO:34. In some embodiments, the nucleicacid sequence encodes an amino acid sequence of SEQ ID NO:4 or anypercent identity thereof described herein.

In some embodiments of the present invention, ACoAS can be provided forproduction and/or accumulation of ACoAS in a heterologous host as wellas for improved production and/or accumulation of ACoAS in an endogenoushost.

In some embodiments, a gene and/or polypeptide encoding ACoAS can beused to identify another ACoAS gene and/or polypeptide sequences and/orcan be used to identify an ACoAS homolog in other cells. Such ACoASencoding sequences can be identified, for example, in the literatureand/or in bioinformatics databases well known to the skilled person. Forexample, the identification of a ACoAS encoding sequence in another celltype using bioinformatics can be accomplished through BLAST (asdisclosed above) searching of publicly available databases with a knownACoAS encoding DNA and polypeptide sequence, such as any of thoseprovided herein. Identities are based on the Clustal W method ofalignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix.

In some embodiments, the genetically modified plant (e.g., soybean),descendant, cell, tissue, or part thereof comprises a PUFA synthase anda ACoAS, or a PUFA synthase, a PPTase and a ACoAS. In some embodiments,the genetically modified plant (e.g., soybean), descendant, cell,tissue, or part thereof comprises the nucleic acid sequences of (i),(ii) or (iii), or any combinations thereof, contained in a singlerecombinant expression vector. In some embodiments, the nucleic acidsequences of (i), (ii) and (iii) are contained in different recombinantexpression vectors. In some embodiments, the nucleic acid sequences of(i) and (ii) are contained in a single recombinant expression vector andthe nucleic acid sequence of (iii) is contained in a differentrecombinant expression vector. In some embodiments, the nucleic acidsequences of (i) and (iii) are contained in a single recombinantexpression vector and the nucleic acid sequence of (ii) is contained ina different recombinant expression vector. In some embodiments, thenucleic acid sequences of (ii) and (iii) are contained in a singlerecombinant expression vector and the nucleic acid sequence of (i) iscontained in a different recombinant expression vector. In someembodiments, the nucleic acid sequences of (i), (ii) or (iii), or anycombinations thereof, are under the control of one or more seed-specificpromoters.

Methods of Making Genetically Modified Organisms

To produce significantly high yields of one or more desiredpolyunsaturated fatty acids, a plant can be genetically modified tointroduce a PUFA synthase into the plant. The present invention alsorelates to methods to improve or enhance the effectiveness of suchgenetic modification and particularly, to improve or enhance theproduction and/or accumulation of the endproduct of a PUFA synthase,e.g., PUFAs.

Methods for gene expression in a genetically modified organism,including, but not limited to plants, are known in the art. In someembodiments, the coding region for the PUFA synthase genes to beexpressed can be codon optimized for the target host cell as describedbelow. Expression of genes in recombinant host cells including, but notlimited to, plant cells, can require a promoter operably linked to acoding region of interest, and/or a transcriptional terminator. A numberof promoters can be used in constructing vectors for genes, includingbut not limited to a seed-specific promoter (e.g., PvDlec2, LfKCS3, FAE1, BoACP, or BnaNapinC) or a leaf-specific promoter (e.g., ubiquitin orCsVMV). Other non-limiting examples of promoters that can be used in thepresent invention include the acyl carrier protein promoter disclosed inWO 1992/18634; the Phaseolus vulgaris beta-phaseolin promoter andtruncated versions disclosed in Slightom et al. (Proc. Natl. Acad. Sci.U.S.A. 80: 1897-1901; 1983); Sengupta-Gopalan et al. (Proc. Nat. Acad.Sci. 82: 3320-3324; 1985); van der Geest et al. (Plant Mol. Biol. 33:553-557; 1997), and Bustos et al. (EMBO J. 10: 1469-1479; 1991).

In some embodiments of the present invention, a recombinant vector is anengineered (e.g., artificially produced) nucleic acid molecule that isused as a tool for manipulating a nucleic acid sequence of choice andfor introducing such a nucleic acid sequence into a host cell. Therecombinant vector is therefore suitable for use in cloning, sequencing,and/or otherwise manipulating the nucleic acid sequence of choice, suchas by expressing and/or delivering the nucleic acid sequence of choiceinto a host cell to form a recombinant cell. Such a vector typicallycontains heterologous nucleic acid sequences, that is nucleic acidsequences that are not naturally found adjacent to nucleic acid sequenceto be cloned or delivered, although the vector can also containregulatory nucleic acid sequences (e.g., promoters, untranslatedregions) that are naturally found adjacent to nucleic acid molecules ofthe present invention or that are useful for expression of the nucleicacid molecules of the present invention. The vector can be either RNA orDNA, either prokaryotic or eukaryotic, and typically is a plasmid. Thevector can be maintained as an extrachromosomal element (e.g., aplasmid) or it can be integrated into the chromosome of a recombinantorganism (e.g., a microbe or a plant). The entire vector can remain inplace within a host cell, or under certain conditions, the plasmid DNAcan be deleted, leaving behind the nucleic acid molecule of the presentinvention. The integrated nucleic acid molecule can be under chromosomalpromoter control, under native or plasmid promoter control, or under acombination of several promoter controls. Single or multiple copies ofthe nucleic acid molecule can be integrated into the chromosome. Arecombinant vector of the present invention can contain at least oneselectable marker.

In some embodiments, a recombinant vector used in a recombinant nucleicacid molecule of the present invention is an expression vector. In suchembodiments, a nucleic acid sequence encoding the product to be produced(e.g., a PUFA synthase) is inserted into the recombinant vector toproduce a recombinant nucleic acid molecule. The nucleic acid sequenceencoding the protein to be produced is inserted into the vector in amanner that operatively links the nucleic acid sequence to regulatorysequences in the vector that enable the transcription and translation ofthe nucleic acid sequence within the recombinant host cell.

Vectors useful for the transformation of a variety of host organisms andcells are common and disclosed in the literature. Typically the vectorcontains a selectable marker and sequences allowing autonomousreplication or chromosomal integration in the desired host. In addition,suitable vectors can comprise a promoter region that harborstranscriptional initiation controls and a transcriptional terminationcontrol region, between which a coding region DNA fragment can beinserted, to provide expression of the inserted coding region. Bothcontrol regions can be derived from genes homologous to the transformedhost cell, although it is to be understood that such control regions canalso be derived from genes that are not native to the specific specieschosen as a production host.

The present invention includes the expression of one or more acyl-CoAsynthetases as described and exemplified herein with a PUFA synthase asdescribed herein and with an exogenous PPTase that are utilized alone orin combination with any one or more strategies described herein (e.g.,any one, two, three, or four of: codon optimization,organelle-targeting, enhancement of PUFA synthase competition formalonyl CoA (e.g., by inhibition of FAS), and/or expression of one ormore acyltransferases or related enzymes), to increase PUFA productionand/or accumulation in a heterologous host.

Some embodiments of the invention relate to the targeting of expressionof the PUFA synthase enzymes, the PPTase, and/or any one or more of theaccessory proteins and/or targeted genetic modifications to one or moreorganelles of the host. For example, in some embodiments, expression ofthe PUFA synthase system and the PPTase can be targeted to the plastidof a plant. In some embodiments, expression of the PUFA synthase and thePPTase is targeted to the cytosol. In some embodiments, expression ofthe PUFA synthase and the PPTase is targeted to both the plastid and thecytosol of a plant. In any of these embodiments, other targets can bedirected to the plastid or the cytosol.

In some embodiments, acyl-CoA synthetases are expressed in the cytosolto convert the DHA and/or other LC-PUFA free fatty acids to acyl-CoAs,which in turn can be utilized by the acyltransferases.

A variety of plastid targeting sequences are known in the art and can beused in embodiments where the heterologous host is a plant or plantcell, and wherein targeting to the plastid is desired.

The present invention includes the use of organelle targeting (e.g., tothe plastid or chloroplast in plants) with expression of a PUFA synthaseas described herein and with an exogenous PPTase, which are utilizedalone or in combination with any one or more strategies described herein(e.g., any one, two, three, or four of: codon optimization, enhancementof PUFA synthase competition for malonyl CoA (e.g., by inhibition ofFAS), expression of one or more acyl-CoA synthetases, and/or expressionof one or more acyltransferases or related enzymes), to increase PUFAproduction and/or accumulation in a heterologous host.

The targeting of gene products to the plastid or chloroplast iscontrolled by a signal sequence found at the amino terminal end ofvarious proteins, which is cleaved during import yielding the matureprotein (e.g., with regard to chloroplast targeting, see, e.g., Comai etal., J. Biol. Chem. 263:15104-15109 (1988)). These signal sequences canbe fused to heterologous gene products to effect the import ofheterologous products into the chloroplast (van den Broeck et al. Nature313:358-363 (1985)). DNA encoding for appropriate signal sequences canbe isolated from the cDNAs encoding the RUBISCO protein, the CABprotein, the EPSP synthase enzyme, the GS2 protein and many otherproteins that are known to be chloroplast localized.

In some embodiments of the invention, the localization of proteinsemployed in the invention is directed to a subcellular compartment, forexample, to the plastid or chloroplast. Proteins can be directed to thechloroplast by including at their amino-terminus a chloroplast transitpeptide (CTP). Similarly, proteins can be directed to the plastid byincluding at their N-terminus a plastid transit or signaling peptide.

Naturally occurring chloroplast targeted proteins, synthesized as largerprecursor proteins containing an amino-terminal chloroplast targetingpeptide directing the precursor to the chloroplast import machinery, arewell known in the art. Chloroplast targeting peptides are generallycleaved by specific endoproteases located within the chloroplastorganelle, thus releasing the targeted mature and can active enzyme fromthe precursor into the chloroplast milieu. Examples of sequencesencoding peptides that are suitable for directing the targeting of thegene or gene product to the chloroplast or plastid of the plant cellinclude the petunia EPSPS CTP, the Arabidopsis EPSPS CTP2 and intron,and others known to those skilled in the art. Such targeting sequencesprovide for the desired expressed protein to be transferred to the cellstructure in which it most effectively functions, or by transferring thedesired expressed protein to areas of the cell in which cellularprocesses necessary for desired phenotypic function are concentrated.Specific examples of chloroplast targeting peptides are well known inthe art and include the Arabidopsis thaliana ribulose bisphosphatecarboxylase small subunit ats1A transit peptide, an Arabidopsis thalianaEPSPS transit peptide, and a Zea maize ribulose bisphosphate carboxylasesmall subunit transit peptide.

An optimized transit peptide is described, for example, by van denBroeck et al., Nature, 313:358-363 (1985). Prokaryotic and eukaryoticsignal sequences are disclosed, for example, by Michaelis et al., Ann.Rev. Microbiol. 36:425 (1982). Additional examples of transit peptidesthat can be used in the invention include the chloroplast transitpeptides such as those described in Von Heijne et al., Plant Mol. Biol.Rep. 9:104-126 (1991); Mazur et al., Plant Physiol. 85:1110 (1987); andVorst et al., Gene 65:59 (1988). Chen & Jagendorf (J. Biol. Chem.268:2363-2367 (1993)) have described use of a chloroplast transitpeptide for import of a heterologous transgene. This peptide used is thetransit peptide from the rbcS gene from Nicotiana plumbaginifolia(Poulsen et al., Mol. Gen. Genet. 205: 193-200 (1986)). One CTP that hasfunctioned herein to localize heterologous proteins to the chloroplastwas derived from Brassica napus acyl-ACP thioesterase.

An alternative means for localizing genes to chloroplast or plastidincludes chloroplast or plastid transformation. Recombinant plants canbe produced in which only the chloroplast DNA has been altered toincorporate the molecules envisioned in this application. Promoters thatfunction in chloroplasts are known in the art (Hanley-Bowden et al.,Trends in Biochemical Sciences 12:67-70 (1987)). Methods andcompositions for obtaining cells containing chloroplasts into whichheterologous DNA has been inserted have been described, for example, byDaniell et al. (U.S. Pat. No. 5,693,507) and Maliga et al. (U.S. Pat.No. 5,451,513).

Combinations of Strategies

According to the present invention, in the production of a heterologoushost for the production and accumulation of one or more target PUFAs,any one or more (any combination) of the strategies described herein forimproving the production and/or accumulation of PUFAs in the host can beused. Indeed, it is anticipated that various combinations of strategieswill be additive or synergistic and provide improved production and/oraccumulation of PUFAs as compared to in the absence of one or more suchstrategies. Indeed, the Examples provide exemplary strategies for theproduction of PUFAs in a host organism.

Any plant or plant cell using these combinations of modifications, orany other modification or combination of modifications described herein,is encompassed by the invention. In some embodiments, such a plant hasbeen further genetically modified to express an accessory protein asdescribed herein for the improvement of the production and/oraccumulation of PUFAs (or other bioactive products of the PUFA synthase)by the host (e.g., ACoAS, GPAT, LPAAT, DAGAT or acetyl CoA carboxylase(ACCase)). Furthermore, any host cell or organism using anymodifications or combination of modifications described herein isencompassed by the invention, as are any products derived from such cellor organisms, including seed or oil comprising the target PUFAs.

In some embodiments, plants to genetically modify according to thepresent invention (e.g., plant host cells) includes, but is not limitedto, any higher plants, including both dicotyledonous andmonocotyledonous plants, and particularly consumable plants, includingcrop plants and especially plants used for their oils. Such plants caninclude, but are not limited to, for example: soybean, rapeseed,linseed, corn, safflowers, sunflowers and tobacco. Thus, any plantspecies or plant cell can be selected. In some embodiments, the plant isof the family Fabaceae (Leguminosae, legume family, pea family, beanfamily or pulse family). In some embodiments, the plant is of the genusGlycine. In some embodiments, the plant is Glycine albicans, Glycineaphyonota, Glycine arenari, Glycine argyrea, Glycine canescens, Glycineclandestine, Glycine curvata, Glycine cyrtoloba, Glycine falcate,Glycine gracei, Glycine hirticaulis, Glycine hirticaulis subsp. leptosa,Glycine lactovirens, Glycine latifolia, Glycine latrobeana, Glycinemicrophylla, Glycine montis-douglas, Glycine peratosa, Glycinepescadrensis, Glycine pindanica, Gycine pullenii, Glycine rubiginosa,Glycine stenophita, Glycine syndetika, Glycine tabacina, Glycinetomentella, Glycine soja, or Glycine max (soybean). In some embodiments,the plant is peanut, beans (Phaseolus vulgaris), broad beans (Viciafaba) or peas (Pisum sativum).

“Plant parts,” as used herein, include any parts of a plant, including,but not limited to, seeds (including mature seeds and immature seeds),pollen, embryos, flowers, fruits, shoots, leaves, roots, stems,explants, etc. In some embodiments, a genetically modified plant has agenome that is modified (e.g., mutated or changed) from its normal(e.g., wild-type or naturally occurring) form such that the desiredresult is achieved (e.g., increased or modified PUFA synthase and/orproduction and/or accumulation of a desired product using the PUFAsynthase). In some embodiments, genetic modification of a plant can beaccomplished using classical strain development and/or molecular genetictechniques. Methods for producing a transgenic plant, wherein arecombinant nucleic acid molecule encoding a desired amino acid sequenceis incorporated into the genome of the plant, are known in the art. Insome embodiments, a plant to genetically modify according to the presentinvention is a plant suitable for consumption by animals, includinghumans.

Plant lines from these plants, optimized for a particularly desirabletrait, e.g., disease resistance, ease of plant transformation, oilcontent or profile, etc., can be produced, selected or identified. Insome embodiments, plant lines can be selected through plant breeding, orthrough methods such as marker assisted breeding and tilling. In someembodiments, plant cell cultures can be used and, for example, are notgrown into differentiated plants and cultivated using ordinaryagricultural practices, but instead grown and maintained in a liquidmedium.

In some embodiments, the plant can be an oil seed plant, wherein the oilseeds, and/or the oil in the oil seeds contain PUFAs produced by thePUFA synthase. In some embodiments, such oils can contain a detectableamount of at least one target or primary PUFA that is the product of thePUFA synthase. In some embodiments, such oils can be substantially freeof intermediate or side products that are not the target or primary PUFAproducts and that are not naturally produced by the endogenous FASsystem in the wild-type plants (e.g., wild-type plants produce someshorter or medium chain PUFAs, such as 18 carbon PUFAs, via the FASsystem, but there will be new, or additional, fatty acids produced inthe plant as a result of genetic modification with a PUFA synthase).

With regard to the production of genetically modified plants, methodsfor the genetic engineering of plants are well known in the art. Forinstance, numerous methods for plant transformation have been developed,including biological and physical transformation protocols fordicotyledonous plants as well as monocotyledonous plants (e.g.,Goto-Fumiyuki et al., Nature Biotech 17:282-286 (1999); and Miki et al.,Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J.E. Thompson, Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993)). Inaddition, vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available, for example, inGruber et al., Methods in Plant Molecular Biology and Biotechnology, B.R. Glick and J. E. Thompson, Eds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

The present invention is drawn to an isolated nucleic acid moleculecomprising a nucleic acid sequence selected from SEQ ID NOs: 6-10 aswell as an isolated nucleic acid molecule comprising a modification ormutation of such a sequence as described herein. The present inventionis draw to isolated polypeptides comprising an amino acid sequenceselected from SEQ ID NOs: 1-5 as well as an isolated polypeptidecomprising a modification or mutation or such a sequence as describedherein.

The present invention includes a recombinant expression vector pDAB7361.The present invention includes a recombinant expression vector pDAB7362.The present invention includes a recombinant expression vector pDAB7363.The present invention includes a recombinant expression vector pDAB7365.The present invention includes a recombinant expression vector pDAB7368.The present invention includes a recombinant expression vector pDAB7369.The present invention includes a recombinant expression vector pDAB7370.The present invention includes a recombinant expression vectorpDAB100518. The present invention includes a recombinant expressionvector pDAB101476. The present invention includes a recombinantexpression vector pDAB9166. The present invention includes a recombinantexpression vector pDAB9167. The present invention includes a recombinantexpression vector pDAB7379. The present invention includes a recombinantexpression vector pDAB7380. The present invention includes a recombinantexpression vector pDAB9323. The present invention includes a recombinantexpression vector pDAB9330. The present invention includes a recombinantexpression vector pDAB9337. The present invention includes a recombinantexpression vector pDAB9338. The present invention includes a recombinantexpression vector pDAB9344. The present invention includes a recombinantexpression vector pDAB9396. The present invention includes a recombinantexpression vector pDAB101412. The present invention includes arecombinant expression vector pDAB7733. The present invention includes arecombinant expression vector pDAB7734. The present invention includes arecombinant expression vector pDAB101493. The present invention includesa recombinant expression vector pDAB109507. The present inventionincludes a recombinant expression vector pDAB109508. The presentinvention includes a recombinant expression vector pDAB109509. Thepresent invention includes a recombinant expression vector pDAB9151. Thepresent invention includes a recombinant expression vector pDAB108207.The present invention includes a recombinant expression vectorpDAB108208. The present invention includes a recombinant expressionvector pDAB108209. The present invention includes a recombinantexpression vector pDAB9159. The present invention includes a recombinantexpression vector pDAB9147. The present invention includes a recombinantexpression vector pDAB108224. The present invention includes arecombinant expression vector pDAB108225.

The present invention includes a soybean plant, descendant, cell,tissue, seed, or part thereof comprising a recombinant expression vectorpDAB7361. The present invention includes a soybean plant, descendant,cell, tissue, seed, or part thereof comprising a recombinant expressionvector pDAB7362. The present invention includes a soybean plant,descendant, cell, tissue, seed, or part thereof comprising a recombinantexpression vector pDAB7363. The present invention includes a soybeanplant, descendant, cell, tissue, seed, or part thereof comprising arecombinant expression vector pDAB7365. The present invention includes asoybean plant, descendant, cell, tissue, seed, or part thereofcomprising a recombinant expression vector pDAB7368. The presentinvention includes a soybean plant, descendant, cell, tissue, seed, orpart thereof comprising a recombinant expression vector pDAB7369. Thepresent invention includes a soybean plant, descendant, cell, tissue,seed, or part thereof comprising a recombinant expression vectorpDAB7370. The present invention includes a soybean plant, descendant,cell, tissue, seed, or part thereof comprising a recombinant expressionvector pDAB100518. The present invention includes a soybean plant,descendant, cell, tissue, seed, or part thereof comprising a recombinantexpression vector pDAB101476. The present invention includes a soybeanplant, descendant, cell, tissue, seed, or part thereof comprising arecombinant expression vector pDAB9166. The present invention includes asoybean plant, descendant, cell, tissue, seed, or part thereofcomprising a recombinant expression vector pDAB9167.

The present invention includes a soybean plant, descendant, cell,tissue, seed, or part thereof comprising a recombinant expression vectorpDAB7379. The present invention includes a soybean plant, descendant,cell, tissue, seed, or part thereof comprising a recombinant expressionvector pDAB7380. The present invention includes a soybean plant,descendant, cell, tissue, seed, or part thereof comprising a recombinantexpression vector pDAB9323. The present invention includes a soybeanplant, descendant, cell, tissue, seed, or part thereof comprising arecombinant expression vector pDAB9330. The present invention includes asoybean plant, descendant, cell, tissue, seed, or part thereofcomprising a recombinant expression vector pDAB9337. The presentinvention includes a soybean plant, descendant, cell, tissue, seed, orpart thereof comprising a recombinant expression vector pDAB9338. Thepresent invention includes a soybean plant, descendant, cell, tissue,seed, or part thereof comprising a recombinant expression vectorpDAB9344. The present invention includes a soybean plant, descendant,cell, tissue, seed, or part thereof comprising a recombinant expressionvector pDAB9396. The present invention includes a soybean plant,descendant, cell, tissue, seed, or part thereof comprising a recombinantexpression vector pDAB101412. The present invention includes a soybeanplant, descendant, cell, tissue, seed, or part thereof comprising arecombinant expression vector pDAB7733. The present invention includes asoybean plant, descendant, cell, tissue, seed, or part thereofcomprising a recombinant expression vector pDAB7734.

The present invention includes a soybean plant, descendant, cell,tissue, seed, or part thereof comprising a recombinant expression vectorpDAB101493. The present invention includes a soybean plant, descendant,cell, tissue, seed, or part thereof comprising a recombinant expressionvector pDAB109507. The present invention includes a soybean plant,descendant, cell, tissue, seed, or part thereof comprising a recombinantexpression vector pDAB109508. The present invention includes a soybeanplant, descendant, cell, tissue, seed, or part thereof comprising arecombinant expression vector pDAB109509. The present invention includesa soybean plant, descendant, cell, tissue, seed, or part thereofcomprising a recombinant expression vector pDAB9151. The presentinvention includes a soybean plant, descendant, cell, tissue, seed, orpart thereof comprising a recombinant expression vector pDAB108207. Thepresent invention includes a soybean plant, descendant, cell, tissue,seed, or part thereof comprising a recombinant expression vectorpDAB108208. The present invention includes a soybean plant, descendant,cell, tissue, seed, or part thereof comprising a recombinant expressionvector pDAB108209. The present invention includes a soybean plant,descendant, cell, tissue, seed, or part thereof comprising a recombinantexpression vector pDAB9159. The present invention includes a soybeanplant, descendant, cell, tissue, seed, or part thereof comprising arecombinant expression vector pDAB9147. The present invention includes asoybean plant, descendant, cell, tissue, seed, or part thereofcomprising a recombinant expression vector pDAB108224. The presentinvention includes a soybean plant, descendant, cell, tissue, seed, orpart thereof comprising a recombinant expression vector pDAB 108225.

As used herein, the term “transfection” is used to refer to any methodby which an exogenous nucleic acid molecule (e.g., a recombinant nucleicacid molecule) can be inserted into a cell. The term “transformation”can be used interchangeably with the term “transfection” when such termis used to refer to the introduction of nucleic acid molecules intomicrobial cells, such as algae, bacteria and yeast, or into plant cells.In microbial and plant systems, the term “transformation” is used todescribe an inherited change due to the acquisition of exogenous nucleicacids by the microorganism or plant and is essentially synonymous withthe term “transfection.” In some embodiments, transfection techniquesinclude, but are not limited to, transformation, particle bombardment,diffusion, active transport, bath sonication, electroporation,microinjection, lipofection, adsorption, infection and protoplastfusion.

A widely utilized method for introducing an expression vector intoplants is based on the natural transformation system of Agrobacterium.Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria known to be useful to geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. C. I. Kado, Crit. Rev. Plant. Sci. 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are also available, e.g., Gruber etal., supra, Miki et al., supra, Moloney et al., Plant Cell Reports 8:238(1989), and U.S. Pat. Nos. 4,940,838 and 5,464,763.

Another known method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface ofmicroprojectiles. In this method, the expression vector is introducedinto plant tissues with a biolistic device that accelerates themicroprojectiles to speeds sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), J. C.Sanford, Trends Biotech. 6:299 (1988), J. C. Sanford, Physiol. Plant79:206 (1990), Klein et al., Biotechnology 10:268 (1992).

Yet another method for physical delivery of DNA to plants is sonicationof target cells. Zhang et al., Bio/Technology 9:996 (1991). Also,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc. Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues has also beendescribed. Donn et al., Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). Additionally, silicone carbide whiskers (Kaepler etal., 1990, Plant Cell Reports) and in plant transformation using, forexample, a flower dipping methodology (Clough and Bent, Plant J.16:735-743 (1998)) can also be used. The exact plant transformationmethodology can vary somewhat depending on the plant species selectedand the plant cell type selected for transformation (e.g., seedlingderived cell types such as hypocotyls and cotyledons or embryonictissue).

Following the introduction of the genetic construct into plant cells,plant cells can be grown and upon emergence of differentiating tissuesuch as shoots and roots, mature plants can be generated. In someembodiments, a plurality of plants can be generated. Methodologies forregenerating plants are known to those of ordinary skill in the art andcan be found, for example, in: Plant Cell and Tissue Culture, 1994,Vasil and Thorpe Eds. Kluwer Academic Publishers and in: Plant CellCulture Protocols (Methods in Molecular Biology 1 11, 1999 Hall EdsHumana Press).

In some embodiments, a genetically modified plant described herein canbe cultured in a fermentation medium or grown in a suitable medium suchas soil. In some embodiments, a suitable growth medium for higher plantscan include any growth medium for plants, including, but not limited to,soil, sand, any other particulate media that support root growth (e.g.,vermiculite, perlite, etc.) or hydroponic culture, as well as suitablelight, water and nutritional supplements that optimize the growth of thehigher plant.

It will be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transfectednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within the host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Additionally, thepromoter sequence might be genetically engineered to improve the levelof expression as compared to the native promoter. Recombinant techniquesuseful for controlling the expression of nucleic acid molecules include,but are not limited to, integration of the nucleic acid molecules intoone or more host cell chromosomes, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals(e.g., promoters, operators, enhancers), substitutions ormodifications of translational control signals (e.g., ribosome bindingsites, Shine-Dalgarno sequences), modification of nucleic acid moleculesto correspond to the codon usage of the host cell, and deletion ofsequences that destabilize transcripts.

In some embodiments, a plant can include those plants that are known toproduce compounds used as pharmaceutical agents, flavoring agents,nutraceutical agents, functional food ingredients or cosmetically activeagents or plants that are genetically engineered to produce thesecompounds/agents.

All of these embodiments of the invention apply to the discussion of anyof the genetically modified organisms and methods of producing and usingsuch organisms as described herein.

Products from Genetically Modified Organisms

In some embodiments, a genetically modified organism of the inventionproduces one or more polyunsaturated fatty acids including, but notlimited to, EPA (C20:5, n-3), DHA (C22:6, n-3), DP A (C22:5, n-6 orn-3), ARA (C20:4, n-6), GLA (CI 8:3, n-6), ALA (C18:3, n-3), and/or SDA(C18:4, n-3)), and in some embodiments, one or more longer-chain PUFAs,including, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), DPA(C22:5, n-6 or n-3), or DTA (C22:4, n-6), or any combination thereof. Insome embodiments, a genetically modified plant of the invention producesone or more polyunsaturated fatty acids including, but not limited to,EPA (C20:5, n-3), DHA (C22:6, n-3), and/or DPA (C22:5, n-6 or n-3), orany combination thereof. In some embodiments, a genetically modifiedplant of the invention does not have a high oleic background.

In some embodiments, a genetically modified organism is a plant that hasbeen genetically modified to recombinantly express a PUFA synthase and aPPTase, as described herein. In some embodiments, such a plant has beengenetically modified further to express an accessory protein asdescribed herein for the improvement of the production and/oraccumulation of PUFAs (or other bioactive products of the PUFA synthase)by the host (e.g., ACoAS, GPAT, LPAAT, DAGAT or ACCase).

Some embodiments of the present invention include the production ofpolyunsaturated fatty acids of desired chain length and with desirednumbers of double bonds and, by extension, oil seed and oils obtainedfrom the genetically modified plants described herein (e.g., obtainedfrom the oil or seeds of such plants) comprising these PUFAs. Examplesof PUFAs that can be produced by the present invention include, but arenot limited to, DHA (docosahexaenoic acid (C22:6, n-3)), ARA(eicosatetraenoic acid or arachidonic acid (C20:4, n-6)), DPA(docosapentaenoic acid (C22:5, n-6 or n-3)), and EPA (eicosapentaenoicacid (C20:5, n-3)), and any combinations thereof. The present inventionallows for the production of commercially valuable lipids enriched inone or more desired (target or primary) PUFAs by the development ofgenetically modified plants through the use of a PUFA synthase thatproduces PUFAs.

In some embodiments, a given PUFA synthase derived from a particularorganism will produce particular PUFA(s), such that selection of a PUFAsynthase from a particular organism will result in the production ofspecified target or primary PUFAs. In some embodiments, the ratio of thePUFAs can differ depending on the selection of the particular PUFAsynthase and on how that system responds to the specific conditions inwhich it is expressed. For example, use of a PUFA synthase fromThraustochytrium 23 B (ATCC No. 20892) can also result in the productionof DHA and DPA(n-6) as the target or primary PUFAs; however, in the caseof Thraustochytrium 23B, the ratio of DHA to DPA(n-6) is 10:1 (and canrange from 8:1 to 40:1), whereas in Schizochytrium, the ratio istypically 2.5:1. In some embodiments, a given PUFA synthase can bemodified by intermixing proteins and domains from different PUFAsynthases, or one can modify a domain or protein of a given PUFAsynthase to change the target PUFA product and/or ratios.

In some embodiments, reference to “intermediate products” or “sideproducts” of an enzyme system that produces PUFAs refers to anyproducts, and particularly, fatty acid products, that are produced bythe enzyme system as a result of the production of the target or primaryPUFA(s) of the system, but that are not the primary or target PUFA(s).In some embodiments, intermediate and side products can includenon-target fatty acids that are naturally produced by the wild-typeplant, or by the parent plant used as a recipient for the indicatedgenetic modification, but are now classified as intermediate or sideproducts because they are produced in greater levels as a result of thegenetic modification, as compared to the levels produced by thewild-type plant, or by the parent plant used as a recipient for theindicated genetic modification. In some embodiments, a primary or targetPUFA of one enzyme system can be an intermediate of a different enzymesystem where the primary or target product is a different PUFA. Forexample, when using the standard pathway to produce EPA, fatty acidssuch as GLA, DGLA and SDA are produced as intermediate products insignificant quantities (e.g., U.S. Appl. Pub. No. 2004/0172682).Similarly, and also illustrated by U.S. Appl. Pub. No. 2004/0172682,when using the standard pathway to produce DHA, in addition to the fattyacids mentioned above, ETA and EPA (notably the target PUFA in the firstexample above) can be produced in significant quantities and can bepresent in significantly greater quantities relative to the total fattyacid product than the target PUFA itself.

In some embodiments, to produce significantly high yields of one or moredesired polyunsaturated fatty acids, a plant can be genetically modifiedto introduce a PUFA synthase system into the plant. Plants are not knownto endogenously contain a PUFA synthase, and therefore, the presentinvention represents an opportunity to produce plants with unique fattyacid production capabilities. The present invention provides geneticallyengineered plants to produce one or more PUFAs in the same plant,including, but not limited to, EPA, DHA, DPA (n3 or n6), ARA, GLA, SDAand others, including any combination thereof. The present inventionoffers the ability to create any one of a number of “designer oils” invarious ratios and forms. In some embodiments, the use of a PUFAsynthase from the particular marine organisms described herein canextend the range of PUFA production and successfully produce such PUFAswithin temperature ranges used to grow most crop plants.

In some embodiments, to be “substantially free” of intermediate or sideproducts of the system for synthesizing PUFAs, or to not haveintermediate or side products present in substantial amounts, means thatany intermediate or side product fatty acids (non-target PUFAs) that areproduced in the genetically modified plant (and/or parts of plantsand/or seed oil fraction) as a result of the introduction or presence ofthe enzyme system for producing PUFAs (e.g., that are not produced bythe wild-type plant or the parent plant used as a recipient for theindicated genetic modification), can be present in a quantity that isless than 10% by weight of total fatty acids, less than 9% by weight oftotal fatty acids, less than 8% by weight of total fatty acids, lessthan 7% by weight of total fatty acids, less than 6% by weight of totalfatty acids, less than 5% by weight of total fatty acids, less than 4%by weight of total fatty acids, less than 3% by weight of total fattyacids, less than 2% by weight of total fatty acids, less than 1% byweight of total fatty acids, or less than 0.5% by weight of total fattyacids.

In some embodiments, a genetically modified plant, descendant, cell,tissue, or part thereof of the invention or an oil or seed obtained froma genetically modified plant, descendant, cell, tissue, or part thereofof the invention comprises detectable amounts of DHA (docosahexaenoicacid (C22:6, n-3)), DPA(n-6) (docosapentaenoic acid (C22:5 n-6)) or EPA(eicosapentaenoic acid (C20:5, n-3)). In some embodiments, a geneticallymodified plant, descendant, cell, tissue, or part thereof of theinvention or an oil or seed obtained from a genetically modified plant,descendant, cell, tissue, or part thereof of the invention comprises atleast 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%,at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, atleast 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, atleast 3.5%), at least 4%, at least 4.5%, at least 5%, at least 5.5%, atleast 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, atleast 8.5%, at least 9%, at least 9.5%, at least 10%, at least 10.5%, atleast 1 1%, at least 1 1.5%, at least 12%, at least 12.5%, at least 13%,at least 13.5%, at least 14%, at least 14.5% or at least 15% DHA byweight of total fatty acids. Useful ranges can be selected between anyof these values, for example, 0.01% to 15%, 0.05% to 10% and 1% to 5%DHA by weight of total fatty acids.

In some embodiments, a genetically modified plant, descendant, cell,tissue, or part thereof of the invention or an oil or seed obtained froma genetically modified plant, descendant, cell, tissue, or part thereofof the invention comprises at least 0.01%, at least 0.02%, at least0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%,at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, atleast 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, atleast 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, atleast 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, atleast 7.5%, at least 8%, at least 8.5%, at least 9%, at least 9.5%, orat least 10% EPA by weight of total fatty acids. Useful ranges can beselected between any of these values, for example, 0.01% to 10%, 0.05%to 5% and 0.1% to 5% EPA by weight of total fatty acids.

In some embodiments, a genetically modified plant, descendant, cell,tissue, or part thereof of the invention or an oil or seed obtained froma genetically modified plant, descendant, cell, tissue, or part thereofof the invention comprises at least 0.01%, at least 0.02%, at least0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%,at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, atleast 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, atleast 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, atleast 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, atleast 7.5%, at least 8%, at least 8.5%, at least 9%, at least 9.5%, orat least 10% DPA(n-6) by weight of total fatty acids. Useful ranges canbe selected between any of these values, for example, 0.01% to 10%,0.01% to 5%, 0.01% to 1%, 0.01% to 0.05%, 0.05% to 5% and 0.1% to 5%DPA(n-6) by weight of total fatty acids.

In some embodiments, a genetically modified plant, descendant, cell,tissue, or part thereof of the invention or an oil or seed obtained froma genetically modified plant, descendant, cell, tissue, or part thereofof the invention comprises a ratio of EPA:DHA of at least 1:1, at least1:1.5, at least 1:2, at least 1:2.5, at least 1:3, at least 1:3.5, atleast 1:4, at least 1:4.5, at least 1:5, at least 1:5.5, at least 1:6,at least 1:6.5, at least 1:7, at least 1:7.5, at least 1:8, at least1:8.5, at least 1:9, at least 1:10, at least 1:11, at least 1:12, atleast 1:13, at least 1:14, at least 1:15, at least 1:16, at least 1:17,at least 1:18, at least 1:19, at least 1:20, at least 1:21, at least1:22, at least 1:23, at least 1:24, at least 1:25, at least 1:26, atleast 1:27, at least 1:28, at least 1:29, or at least 1:30 by weight oftotal fatty acids. Useful ranges can be selected between any of thesevalues, for example, a ratio of EPA:DHA of 1:1 to 1:30, 1:1 to 1:25, 1:1to 1:20, 1:1 to 1:15, 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:3, and 1:1 to1:2 by weight of total fatty acids.

In some embodiments, a genetically modified plant, descendant, cell,tissue, or part thereof of the invention or an oil or seed obtained froma genetically modified plant, descendant, cell, tissue, or part thereofof the invention comprises a ratio of DPA(n-6):DHA of at least 1:1, atleast 1:1.5, at least 1:2, at least 1:2.5, at least 1:3, at least 1:3.5,at least 1:4, at least 1:4.5, at least 1:5, at least 1:5.5, at least1:6, at least 1:6.5, at least 1:7, at least 1:7.5, at least 1:8, atleast 1:8.5, at least 1:9, or at least 1:10 by weight of total fattyacids. Useful ranges can be selected between any of these values, forexample, a ratio of DPA(n-6):DHA of 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:3and 1:1 to 1:2 by weight of total fatty acids.

In some embodiments, an oil obtained from a genetically modified plant,descendant, cell, tissue, or part thereof or seed of the inventioncomprises at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% triglycerides by weight of theoil. In some embodiments, an oil obtained from a genetically modifiedplant, descendant, cell, tissue, or part thereof or seed of theinvention comprises from 70% to 99% triglycerides by weight of the oil,from 75% to 99% triglycerides by weight of the oil, from 80%) to 99%triglycerides by weight of the oil, from 85% to 99% triglycerides byweight of the oil, or from 90% to 99% triglycerides by weight of theoil. Methods for purification and analysis of triglyceride have beendescribed (e.g., V. Ruiz-Gutierrez and L. J. Barron, J. Chromatogr. B.Biomed. Appl., 671:133-168, 1995).

In some embodiments, when the target product of a PUFA synthase systemis a long chain PUFA, such as DHA, DP A (n-6 or n-3), or EPA,intermediate products and side products that are not present insubstantial amounts in the total lipids of plants genetically modifiedwith such a PUFA synthase system can include, but are not limited to:gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA;18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6),arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9)and various other intermediate or side products, such as 20:0; 20:1(Δ5); 20:1 (Δ11); 20:2 (Δ8,11); 20:2 (Δ11,14); 20:3 (Δ5,11,14); 20:3(Δ11,14,17); mead acid (20:3; Δ5,8,11); or 20:4 (Δ5,1,14,17).

The genetic modification of a plant according to the present inventioncan result in the production of one or more PUFAs by the plant. In someembodiments, the PUFA profile and the ratio of the PUFAs produced by theplant are not necessarily the same as the PUFA profile or ratio of PUFAsproduced by the organism from which the PUFA synthase was derived.

In some embodiments, a genetically modified plant, descendant, cell,tissue, or part thereof of the present invention can be engineered toproduce PUFAs through the activity of the PUFA synthase. In someembodiments, the PUFAs can be recovered through purification processesthat extract the compounds from the plant, descendant, cell, tissue, orpart thereof. In some embodiments, the PUFAs can be recovered byharvesting the plant, descendant, cell, tissue, or part thereof In someembodiments, the PUFAs can be recovered by harvesting the oil from theplant, descendant, cell, tissue, or part thereof (e.g., from the oilseeds) or seeds from the plant, descendant, cell, tissue, or partthereof. In some embodiments, the plant, descendant, cell, tissue, orpart thereof can also be consumed in its natural state or furtherprocessed into consumable products.

In some embodiments, a genetically modified plant, descendant, cell,tissue, or part thereof of the invention can produce one or morepolyunsaturated fatty acids. In some embodiments, the plant, descendant,cell, tissue, or part thereof can produce (e.g., in its mature seeds, ifan oil seed plant, or in the oil of the seeds of an oil seed plant) atleast one PUFA (the target PUFA), and wherein the total fatty acidprofile in the plant, or the part of the plant that accumulates PUFAs(e.g., mature seeds, if the plant is an oil seed plant or the oil of theseeds of an oil seed plant), comprises a detectable amount of this PUFAor PUFAs. In some embodiments, the target PUFA is at least a 20 carbonPUFA and comprises at least 3 double bonds, at least 4 double bonds, orat least 5 double bonds. In some embodiments, the target PUFA can be aPUFA that is not naturally produced by the plant. In some embodiments,the total fatty acid profile in the plant or in the part of the plantthat accumulates PUFAs (including the seed oil of the plant) comprisesat least 0.1% of the target PUFA(s) by weight of total fatty acids, atleast 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 1%, atleast 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, atleast 4%, at least 4.5%, at least 5%, at least 5.5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, more than 75% of at least onepolyunsaturated fatty acid (the target PUFA or PUFAs) by weight of totalfatty acids, or any percentage from 0.1% to 75%, or greater than 75% (upto 100% or 100%), in 0.1% increments, of the target PUFA(s).

As used herein, reference to a percentage amount of PUFA is thepercentage by weight of total fatty acids extracted, unless otherwisestated. In some embodiments, total fatty acids are determined by gaschromatography (GC) analysis of a fatty acid methyl ester (F AME)preparation, although determination of total fatty acids is not limitedto this method.

In some embodiments, the total fatty acids in a plant of the invention(and/or descendant, cell, tissue, or part thereof or seed oil fraction)can contain less than 10% by weight of the total fatty acids produced bythe plant, less than 9% by weight of the total fatty acids produced bythe plant, less than 8% by weight of the total fatty acids produced bythe plant, descendant, cell, tissue, or part thereof, less than 7% byweight of the total fatty acids produced by the plant, descendant, cell,tissue, or part thereof, less than 6% by weight of the total fatty acidsproduced by the plant, descendant, cell, tissue, or part thereof, lessthan 5% by weight of the total fatty acids produced by the plant,descendant, cell, tissue, or part thereof, less than 4% by weight of thetotal fatty acids produced by the plant, descendant, cell, tissue, orpart thereof, less than 3% by weight of the total fatty acids producedby the plant, descendant, cell, tissue, or part thereof, less than 2% byweight of the total fatty acids produced by the plant, descendant, cell,tissue, or part thereof, less than 1% by weight of the total fatty acidsproduced by the plant, descendant, cell, tissue, or part thereof of afatty acid selected from gamma-linolenic acid (GLA; 18:3, n-6);stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic acid(DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6);eicosatrienoic acid (ETA; 20:3, n-9) and various other fatty acids, suchas 20:0; 20:1 (Δ5); 20:1 (Δ11); 20:2 (Δ8,11); 20:2 (Δ11,14); 20:3(A5,11,14); 20:3 (Δ11,14,17); mead acid (20:3; Δ5,8,11); or 20:4(Δ5,1,14,17).

The present invention includes any seed produced by the plants,descendants, cells, tissues, or parts thereof described herein, as wellas any oil produced by a plant, descendant, cell, tissue, or partthereof or seed of the present invention. The present invention alsoincludes any products produced using the plants, descendants, cells,tissues, or parts thereof, seed or oils as described herein.

Uses and Products Related to the Genetically Modified Organisms of theInvention

The present invention includes a method to produce PUFAs by growing orculturing a genetically modified plant, descendant, cell, tissue, orpart thereof (e.g., soybean) of the present invention described indetail above. In some embodiments, such a method includes, for example,growing in a suitable environment, such as soil, a plant that has agenetic modification as described previously herein and in accordancewith the present invention.

The present invention includes a method to produce an oil comprising atleast one PUFA, comprising recovering oil from a genetically modifiedplant, descendant, cell, tissue, or part thereof of the invention orfrom a seed of a genetically modified plant, descendant, cell, tissue,or part thereof of the invention.

The present invention includes a method to produce an oil comprising atleast one PUFA, comprising growing a genetically modified plant,descendant, cell, tissue, or part thereof of the invention. The presentinvention includes a method to produce at least one PUFA in a seed oilcomprising recovering an oil from a seed of a genetically modifiedplant, descendant, cell, tissue, or part thereof of the invention. Thepresent invention includes a method to produce at least one PUFA in aseed oil comprising growing a genetically modified plant, descendant,cell, tissue, or part thereof of the invention.

The present invention includes a method to provide a supplement ortherapeutic product containing at least one PUFA to an individual inneed thereof, comprising providing to the individual in need thereof agenetically modified plant, descendant, cell, tissue, or part thereof ofthe invention, an oil of the invention, a seed of the invention, a foodproduct of the invention, a functional food of the invention, or apharmaceutical product of the invention. The present invention alsoincludes a method to produce a genetically modified plant, descendant,cell, tissue, or part thereof of the invention comprising transforming aplant or plant cell with (i) a nucleic acid sequence encoding an algalPUFA synthase system that produces at least one polyunsaturated fattyacid (PUFA); and (ii) a nucleic acid sequence encoding aphosphopantetheinyl transferase (PPTase) that transfers aphosphopantetheinyl cofactor to an algal PUFA synthase system ACPdomain. In some embodiments, the method further comprises transformingthe plant or plant cell with (iii) a nucleic acid sequence encoding anacyl-CoA synthetase (ACoAS) that catalyzes the conversion of long chainPUFA free fatty acids (FF A) to acyl-CoA.

In some embodiments, the PUFA of such methods of the invention is DHA,DPA(n-6) and/or EPA. In some embodiments, the oil produced by suchmethods of the invention is a soybean oil. In some embodiments, the oilproduced by such methods of the invention comprises 0.05% to 15% DHA byweight of total fatty acids, or any amount or range thereof describedfurther herein. In some embodiments, the oil produced by such methods ofthe invention further comprises 0.01% to 5% EPA by weight of total fattyacids, or any amount or range thereof described further herein. In someembodiments, the oil produced by such methods of the invention furthercomprises 0.01% to 5% DPA(n-6) by weight of total fatty acids, or anyamount or range thereof described further herein. In some embodiments,the oil produced by such methods of the invention comprises a ratio ofEPA:DHA of 1:1 to 1:30 by weight of total fatty acids, a ratio ofEPA:DHA of 1:1 to 1:3 by weight of total fatty acids, or any amount orrange thereof described further herein. In some embodiments, the oilproduced by such methods of the invention further comprises a ratio ofDPA(n-6):DHA of 1:1 or 1:10 by weight of total fatty acids, a ratio ofDPA(n-6):DHA of 1:1 to 1:3 by weight of total fatty acids, or any amountor range thereof described further herein.

The present invention further includes any organisms or parts thereofdescribed herein (e.g., plants, descendants, cells, tissues, seeds, orparts thereof (e.g., oil seeds), or preparations or fractions thereof),as well as any oils produced by the organisms described herein. Theinvention also includes any products produced using the organisms, partsthereof, or oils described herein.

The present invention relates to a method to modify a product containingat least one fatty acid, comprising adding to the product an organism,part thereof, or oil produced by a genetically modified organismaccording to the invention and as described herein (e.g., a plant,descendant, cell, seed, tissue, or part thereof that has beengenetically modified as described herein). Any products produced by thismethod or generally containing any organisms, parts thereof, or oilsfrom the organisms described herein are also encompassed by theinvention.

In some embodiments, the product is selected from a food dietarysupplement, a pharmaceutical formulation, a humanized animal milk, aninfant formula, a nutraceutical and a functional food. Suitablepharmaceutical formulations include, but are not limited to, ananti-inflammatory formulation, a chemotherapeutic agent, an activeexcipient, an osteoporosis drug, an anti-depressant, an anti-convulsant,an anti-Helicobacter pylori drug, a drug for treatment ofneurodegenerative disease, a drug for treatment of degenerative liverdisease, an antibiotic, and a cholesterol lowering formulation. In someembodiments, the product is used to treat a condition selected fromchronic inflammation, acute inflammation, gastrointestinal disorder,cancer, cachexia, cardiac restenosis, neurodegenerative disorder,degenerative disorder of the liver, blood lipid disorder, osteoporosis,osteoarthritis, autoimmune disease, preeclampsia, preterm birth, agerelated maculopathy, pulmonary disorder, and peroxisomal disorder.

In some embodiments, the product is a food product or functional foodproduct. Suitable food products include, but are not limited to, finebakery wares, bread and rolls, breakfast cereals, processed andunprocessed cheese, condiments (ketchup, mayonnaise, etc.), dairyproducts (milk, yogurt), puddings and gelatin desserts, carbonateddrinks, teas, powdered beverage mixes, processed fish products,fruit-based drinks, chewing gum, hard confectionery, frozen dairyproducts, processed meat products, nut and nut-based spreads, pasta,processed poultry products, gravies and sauces, potato chips and otherchips or crisps, chocolate and other confectionery, soups and soupmixes, soya based products (e.g., milks, drinks, creams, whiteners),vegetable oil-based spreads, and vegetable-based drinks.

In some embodiments of the invention, the product is a feed or mealcomposition, or an additive for a feed or meal composition, for ananimal. The term “animal” includes humans and non-humans. Non-limitingexamples of animals are non-ruminants (e.g., pigs, poultry, or fish),and ruminants (e.g., cows, sheep and horses). The term feed or feedcomposition means any compound, preparation, mixture, or compositionsuitable for, or intended for intake by an animal.

In some embodiments, the invention is directed to an oil blendcomprising an oil obtained from a genetically modified plant,descendant, tissue, or part thereof described herein, and another oil.In some embodiments, the another oil is seed oil, vegetable oil, fishoil, microbial oil, or mixture thereof.

In some embodiments, an oil obtained from a genetically modified plant,descendant, tissue, or part thereof described herein can be furtherprocessed to modify the LC-PUFAs in the oil, for example, to form estersand/or to purify the LC-PUFAs for medicinal purposes.

Some embodiments of the present invention are directed to a soybean oilcomprising 0.05% to 15% DHA by weight of total fatty acids, or any rangethereof described further herein. In some embodiments, the soybean oilfurther comprises 0.05% to 5% EPA by weight of total fatty acids. Insome embodiments, the soybean oil further comprises 0.01% to 5% DPA(n-6)by weight of total fatty acids. In some embodiments, the soybean oil hasa fatty acid profile of greater than 3.5% alpha-linolenic acid by weightof total fatty acids or any range thereof described further herein. Someembodiments of the present invention are directed to a compositioncomprising a soybean oil described herein. In some embodiments, thecomposition comprising a soybean oil comprises one or more oils. In someembodiments, the composition does not contain a PUFA (e.g., DHA) from asource that is not soybean.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES Example 1 Codon Optimization of PUFA Synthase OrfA, PUFASynthase OrfB, PUFA Synthase OrfC, Acyl-CoA Synthetase and 4′Phosphopantetheinyl Transferase HetI

Analysis of the DNA sequences encoding PUFA synthase OrfA fromSchizochytrium sp. ATCC 20888 (GenBank ID: AF378327, GI:158518688), PUFAsynthase OrfB from Schizochytrium sp. ATCC 20888 (GenBank ID: AF378328,GI:158518690), PUFA synthase chimeric OrfC from Schizochytrium sp. ATCC20888 and Thraustochytrium (U.S. Appl. Pub. No. 2008/0022422, now U.S.Pat. No. 8,003,772, issued Aug. 23, 2011) (also described as “hybridOrfC”), acyl-CoA synthetase from Schizochytrium sp. ATCC 20888 (U.S.Appl. Pub. No. 2007/0245431), and 4′ phosphopantetheinyl transferaseHetI from Nostoc sp. PCC 7120 (GenBank ID: P37695, GI:20141367) revealedthe presence of several sequence motifs containing non-optimal codoncompositions that may be detrimental to optimal plant expression. Thedesign of the gene(s) encoding PUFA synthase OrfA, PUFA synthase OrfB,PUFA synthase chimeric OrfC, acyl-CoA synthetase and 4′phosphopantetheinyl transferase HetI proteins was optimized to generatea DNA sequence that is more “plant-like” in nature, and in which thesequence modifications do not hinder translation or create mRNAinstability through non-optimal codon composition.

Due to the plasticity afforded by the redundancy/degeneracy of thegenetic code (e.g., some amino acids are specified by more than onecodon), evolution of the genomes in different organisms or classes oforganisms has resulted in differential usage of synonymous codons. This“codon bias” is reflected in the mean base composition of protein codingregions. For example, organisms having genomes with relatively low G+Ccontents utilize more codons having A or T in the third position ofsynonymous codons, whereas those having higher G+C contents utilize morecodons having G or C in the third position. Further, it is thought thatthe presence of “minor” codons within an mRNA may reduce the absolutetranslation rate of that mRNA, especially when the relative abundance ofthe charged tRNA corresponding to the minor codon is low. An extensionof this reasoning is that the diminution of translation rate byindividual minor codons would be at least additive for multiple minorcodons. Therefore, mRNAs having high relative contents of minor codonswould have correspondingly low translation rates. This rate would bereflected by correspondingly low levels of the encoded protein.

In engineering genes encoding a PUFA synthase OrfA, PUFA synthase OrfB,PUFA synthase chimeric OrfC, acyl-CoA synthetase and 4′phosphopantetheinyl transferase HetI protein for expression indicotyledonous plants (such as tobacco, soybean, cotton or canola), thecodon usages for canola were accessed from publicly available databases(Table 1).

TABLE 1Synonymous codon representation in coding regions of dicotyledonousplants from Brassica napus (canola) genes (Columns C and G). Valuesfor a balanced-biased codon representation set for a plant-optimizedsynthetic gene design are in Columns D and H. C D G H A B CanolaWeighted E F Canola Weighted Amino Acid Codon % Average Amino Acid Codon% Average ALA (A) GCA 23.3 23.3 LEU (L) CTA 10.1 DNU GCC 21.2 21.2 CTC22.8 28.5 GCG 14.2 14.2 CTG 11.6 14.6 GCT 41.3 41.3 CTT 25.2 31.6ARG (R) AGA 31.8 43.8 TTA 10.1 DNU AGG 22.1 30.5 TTG 20.2 25.3 CGA 9.9DNU LYS (K) AAA 44.6 44.6 CGC 8.9 DNU AAG 55.4 55.4 CGG 8.6 DNU MET (M)ATG 100.0 100.0 CGT 18.6 25.7 PHE (F) TTC 58.6 58.6 ASN (N) AAC 62.662.6 TTT 41.4 41.4 AAT 37.4 37.4 PRO (P) CCA 29.6 29.6 ASP (D) GAC 42.542.5 CCC 14.6 14.6 GAT 57.5 57.5 CCG 18.4 18.4 CYC (C) TGC 49.2 49.2 CCT37.3 37.3 TGT 50.8 50.8 SER (S) AGC 16.0 17.9 END TAA 38.5 DNU AGT 14.115.8 TAG 22.1 DNU TCA 18.2 20.4 TGA 39.4 100.00 TCC 16.7 18.7 GLN (Q)CAA 50.0 50.0 TCG 10.7 DNU CAG 50.0 50.5 TCT 24.3 27.2 GLU (E) GAA 43.643.6 THR (T) ACA 26.3 26.3 GAG 56.4 56.4 ACC 26.9 26.9 GGA 36.4 36.4 ACG16.9 16.9 GGC 16.2 16.2 ACT 30.0 30.0 GGG 15.2 15.2 TRP (W) TGG 100.0100.0 GGT 32.1 32.1 TYR (Y) TAC 59.4 59.4 HIS (H) CAC 49.6 49.6 TAT 40.640.6 CAT 50.4 50.4 VAL (V) GTA 10.8 DNU ILE (I) ATA 21.1 21.1 GTC 24.127.0 ATC 42.7 42.7 GTG 28.3 31.7 ATT 36.2 36.2 GTT 36.8 41.3 *DNU-DO NotUse

To balance the distribution of the remaining codon choices for an aminoacid, a Weighted Average representation for each codon was calculated(Table 1), using the formula: Weighted Average % of C1=1/(% C1+%C 2+%C3+etc.)×%C1×100, where C1 is the codon in question and % C2, % C3, etc.represent the averages of the % values for canola of remainingsynonymous codons (average % values for the relevant codons are takenfrom Columns C and G) of Table 1. The Weighted Average % value for eachcodon is given in Columns D and H of Table 1.

In designing coding regions for plant expression, the primary (“firstchoice”) codons preferred by the plant was determined, as well as thesecond, third, fourth etc. choices of preferred codons when multiplechoices exist. A new DNA sequence was then designed that encodedessentially the same amino acid sequence of an PUFA synthase OrfA, PUFAsynthase OrfB, PUFA synthase OrfC, acyl-CoA synthetase and 4′phosphopantetheinyl transferase HetI, but that differed from theoriginal DNA sequence (encoding the PUFA synthase OrfA, PUFA synthaseOrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase and 4′phosphopantetheinyl transferase HetI) by the substitution of plant(first preferred, second preferred, third preferred, or fourthpreferred, etc.) codons to specify the amino acid at each positionwithin the amino acid sequence.

The new sequences were then analyzed for restriction enzyme sitescreated by the modifications in the sequence. The identified sites werethen modified by replacing the codons with first, second, third, orfourth choice preferred codons. The sequence was then further analyzedand modified to reduce the frequency of TA or GC doublets.

Analysis of these sequences revealed that the new DNA sequences encodedessentially the amino acid sequence of the PUFA synthase OrfA, PUFAsynthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase and 4′phosphopantetheinyl transferase HetI proteins but were respectivelydesigned for optimal expression in dicotyledonous plants using abalanced codon distribution of frequently used codons found in canolagenes. In particular, the new DNA sequences differed from the originalDNA sequences encoding an PUFA synthase OrfA, PUFA synthase OrfB, PUFAsynthase chimeric OrfC, acyl-CoA synthetase and 4′ phosphopantetheinyltransferase HetI by the substitution of plant (first preferred, secondpreferred, third preferred, or fourth preferred) codons to specify theappropriate amino acid at each position within the protein amino acidsequence.

Design of the plant-optimized DNA sequences were initiated byreverse-translation of the protein sequences of PUFA synthase OrfA (SEQID NO: 1), PUFA synthase OrfB (SEQ ID NO: 2), PUFA synthase chimericOrfC (SEQ ID NO: 3), acyl-CoA synthetase (SEQ ID NO: 4) and 4′phosphopantetheinyl transferase HetI (SEQ ID NO: 5) using a canola codonbias table constructed from Table 1, Columns D and H. The proteinsequence for acyl-CoA synthetase (SEQ ID NO: 4) was altered from theoriginal sequence; wherein the second amino acid Alanine was removedfrom the protein. The initial sequences were then modified bycompensating codon changes (while retaining overall weighted averagecodon representation) to remove or add restriction enzyme recognitionsites, remove highly stable intrastrand secondary structures, and removeother sequences that might be detrimental to cloning manipulations orexpression of the engineered gene in plants. The DNA sequences were thenre-analyzed for restriction enzyme recognition sites that might havebeen created by the modifications. The identified sites were furthermodified by replacing the relevant codons with first, second, third, orfourth choice preferred codons. Other sites in the sequences that couldaffect transcription or translation of the gene of interest include theexon:intron junctions (5′ or 3′), poly A addition signals, or RNApolymerase termination signals. The modified sequences were furtheranalyzed and further modified to reduce the frequency of TA or CGdoublets, and to increase the frequency of TG or CT doublets. Inaddition to these doublets, sequence blocks that have more than aboutsix consecutive residues of [G+C] or [A+T] can affect transcription ortranslation of the sequence. Therefore, these sequence blocks were alsomodified by replacing the codons of first or second choice, etc. withother preferred codons of choice. Rarely used codons are not included toa substantial extent in the gene design, being used only when necessaryto accommodate a different design criterion than codon composition perse (e.g., addition or deletion of restriction enzyme recognition sites).

The protein encoded by PUFA synthase OrfA comprises 10 repeated“Proline-Alanine” domains ranging in size from 17 to 29 amino acids.Interspersed between the Proline-Alanine repeats were 9 longer repeatedsequence domains comprising 87 amino acids. The amino acid sequences ofthese repeats vary at only 4 positions, and there were only two codonchoices at each of the variant positions. Analyses of the amino acidsequences of the 9 repeats using the Clustal W computer programgenerated a homology value of 100%, and an identity value of 95.4%. Atthe DNA level, the sequences encoding the 9 repeats are 100%)homologous, 89.7% identical, varying at only 27 positions in the 261bases encoding each repeat (23 of the 27 changes are “silent”differences, in which synonymous codons for the same amino acid areinterchanged).

Standard gene design processes cannot easily accommodate developing newcodon biased DNA sequences for multiple repeats of this size, since onemust continually balance all the codon choices in an individual repeatwith the codon choices made at the same position in the other 8 repeats,to avoid generating highly related DNA sequences. For each of the 87residue repeats, there were more than 4.5×10⁴³ possible DNA sequences toencode the same amino acid sequence (calculated as the product of thenumber of synonymous codons for each amino acid in the sequence). Thus,there was a very large computing space available to generateidentically-encoding DNA sequences. The following protocol describes amethod used to generate (in silico) multiple sequence designs for eachindividual repeat, followed by comparison of all the sequence versionsin bulk to identify a set that represents highly diverged sequencesencoding the repeats:

Step 1: Extract the native DNA sequence encoding each repeated aminoacid domain as a separate sequence.

Step 2: Import the individual repeated DNA sequences as separatesequences into a gene design program (e.g., OPTGENETM, OcimumBiosolutions, Hyderabad, India). Steps 3-5 are performed on eachsequence separately.

Step 3: Translate the DNA sequence using the standard genetic code.

Step 4: Reverse translate the translated protein sequence using thestandard genetic code and the appropriate codon bias table. In thisexample, a biased codon table compiled from 530 Brassica napus proteincoding regions was used, and each generated sequence was code-named“nap” (for “napus”) plus the version number. Thus, the firstreverse-translated, codon biased sequence for Repeat 1 was named “rptlnap1.” In this illustration, this process was performed 10 times, togenerate 10 DNA sequence versions encoding the protein sequence ofRepeat 1.

Step 5: Export the 10 sequence versions into the corresponding number oftext files.

Step 6: Repeat Steps 3-5 for each of the other repeated sequencedomains. In this illustration, a total of 90 “nap” sequence versionswere generated (10 for each repeated element).

Step 7: Import the 90 sequence files into the Clustal W program Mega 3.1(accessed at Megasoftware) and perform a multiple sequence alignmentusing all 90 sequences as input. Because these sequences are segments ofprotein coding regions, the alignments are performed with no gapsallowed. After Clustal W Alignment, a Neighbor-Joining tree is assembledand visualized, and one of the ten codon-optimized sequences for each ofthe nine repeated domains in the protein is picked visually. Eachselected sequence version is chosen from a section of the tree that isthe most deeply branched.

Step 8: The chosen sequence for each repeated domain is incorporatedinto the codon-optimized DNA sequence encoding the entire protein, inthe proper position for each particular repeat.

Step 9: Final analyses of the entire codon optimized sequence, includingthe separately designed diverged repeat elements, are performed toassure the absence of undesired motifs, restriction enzyme recognitionsites, etc.

Employing this method with the codon optimization of the PUFA synthaseOrfA coding sequence resulted in the selection of repeatedProline-Alanine sequences that are sufficiently diverged to avoidrepeated sequence instability. These sequences were chosen from thedeepest branches of the Neighbor-Joining tree (i.e., are the mostdistantly related to one another in this sequence set). Smith-Wassermanglobal alignments were done for all pair wise combinations and the rangeof homology was 74-81% with a probable median of 76-77% (Table 2).

TABLE 2 Smith-Wasserman homologies of selected codon-optimized sequencesof repeats of PUFA OrfA. rpt1 nap9 rpt2 nap10 rpt3 nap10 rpt4 nap1 rpt5nap 10 rpt6 nap6 rpt7 nap9 rpt8 nap4 rpt9 nap10 rpt1 nap9 100 77 74 7774 77 81 76 76 rpt2 nap10 100 81 76 74 77 79 76 77 rpt3 nap10 100 79 8074 74 76 78 rpt4 nap1 100 80 77 75 76 76 rpt5 nap10 100 78 77 77 77 rpt6nap6 100 78 76 77 rpt7 nap9 100 75 74 rpt8 nap4 100 76 rpt9 nap10 100

A Clustal W alignment (Vector NTI, Invitrogen, Carlsbad, Calif.) of thechosen 9 newly designed coding regions for the 9 repeated domains isshown in FIG. 1. Overall, the sequences are 93.1% homologous, 61.7%identical as compared to the original sequences, which were 100%homologous and 89.7% identical. Greater sequence divergence could beachieved by using more than 10 sequence iterations and employing acomputer program or mathematical algorithm to select from thesesequences (instead of choosing sequences visually). Nevertheless, thesequences exemplified are highly divergent, and produced stablepoly-nucleotide fragments.

The newly designed, canola optimized PUFA synthase OrfA, PUFA synthaseOrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase and 4′phosphopantetheinyl transferase HetI DNA sequences are listed,respectively, in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9and SEQ ID NO: 10. These codon optimized sequences are identified asversion 3 (v3) throughout the specification, whereas the sequences thatare non-codon optimized are referred to as version 2 (v2) throughout thespecification.

The resulting DNA sequences have a higher degree of codon diversity, adesirable base composition, contain strategically placed restrictionenzyme recognition sites, and lack sequences that might interfere withtranscription of the gene, or translation of the product mRNA. Table 3,Table 4, Table 5, Table 6 and Table 7 present the comparisons of thecodon compositions of the coding regions for the PUFA synthase OrfA,PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase and4′ phosphopantetheinyl transferase HetI proteins found in the originalgene, the plant-optimized versions and the codon compositionrecommendations for a plant optimized sequence as calculated from Table1, Columns D and H.

TABLE 3 PUFA OrfA codon compositions Plnt Plnt Plnt Plnt Plnt Plnt AminoOriginal Original Opt Opt Opt Amino Original Original Opt Opt Opt AcidCodon Gene # Gene % Gene # Gene % Recm'd Acid Codon Gene # Gene % Gene #Gene % Recm'd ALA (A) GCA    7 1.5  109 23.3 23.3 LEU (L) CTA    0 0.0   0 0.0 0.0 GCC  302 64.5   99 21.2 21.2 CTC  173 77.9   63 28.4 28.5GCG   49 10.5   67 14.3 14.2 CTG   15 6.8   32 14.4 14.6 GCT  110 23.5 193 41.2 41.3 CTT   33 14.9   71 32.0 31.6 ARG (R) AGA    0 0.0   5743.5 43.8 TTA    0 0.0    0 0.0 0.0 AGG    0 0.0   40 30.5 30.5 TTG    10.5   56 25.2 25.3 CGA    0 0.0    0 0.0 0.0 LYS (K) AAA    2 1.2   7344.5 44.6 CGC  112 85.5    0 0.0 0.0 AAG  162 98.8   91 55.5 55.4 CGG   1 0.8    0 0.0 0.0 MET (M) ATG   88 100   88 100 100.0 CGT   18 13.7  34 26.0 25.7 PHE (F) TTC   50 69.4   42 58.3 58.6 ASN (N) AAC   7397.3   47 62.7 62.6 TTT   22 30.6   30 41.7 41.4 AAT    2 2.7   28 37.337.4 PRO (P) CCA    2 1.3   45 30.0 29.6 ASP (D) GAC  126 76.8   70 42.742.5 CCC   56 37.3   22 14.7 14.6 GAT   38 23.2   94 57.3 57.5 CCG   4630.7   27 18.0 18.4 CYS (C) TGC   34 94.4   18 50.0 49.2 CCT   46 30.7  56 37.3 37.3 TGT    2 5.6   18 50.0 50.8 SER (S) AGC   40 21.3   3418.1 17.9 END TAA    1 100.0    0 0.0 0.0 AGT    1 0.5   30 16.0 15.8TAG    0 0.0    0 0.0 0.0 TCA    0 0.0   38 20.2 20.4 TGA    0 0.0    1100.0 100.0 TCC   70 37.2   35 18.6 18.7 GLN (Q) CAA    4 4.4   46 50.550.0 TCG   59 31.4    0 0.0 0.0 CAG   87 95.6   45 49.5 50.0 TCT   189.6   51 27.1 27.2 GLU (E) GAA    9 3.8  103 43.6 43.6 THR (T) ACA    21.3   41 26.3 26.3 16 GAG  227 96.2  133 56.4 56.4 ACC   81 51.9   4226.9 26.9 GLY (G) GGA    6 3.1   71 36.2 36.4 ACG   26 16.7   26 16.716.9 GGC  156 79.6   32 16.3 16.2 ACT   47 30.1   47 30.1 30.0 GGG    00.0   30 15.3 15.2 TRP (W) TGG   13 100   13 100 100.0 GGT   34 17.3  63 32.1 32.1 TYR (Y) TAC   42 97.7   26 60.5 59.4 HIS (H) CAC   2583.3   15 50.0 49.6 TAT    1 2.3   17 39.5 40.6 CAT    5 16.7   15 50.050.4 VAL (V) GTA    0 0.0    0 0.0 0.0 ILE (I) ATA    0 0.0   29 21.021.1 GTC  176 70.7   67 26.9 27.0 ATC   99 71.7   59 42.8 42.7 GTG   3915.7   79 31.7 31.7 ATT    39 28.3   50 36.2 36.2 GTT   34 13.7  10341.4 41.3 Totals 1566 1566 Totals 1345 1345

TABLE 4 PUFA OrfB codon compositions Plnt Plnt Plnt Plnt Plnt Plnt AminoOriginal Original Opt Opt Opt Amino Original Original Opt Opt Opt AcidCodon Gene # Gene % Gene # Gene % Recm'd Acid Codon Gene # Gene % Gene #Gene % Recm'd ALA (A) GCA   13 5.7   53 23.2 23.3 LEU (L) CTA   0 0.0  0 0.0 0.0 GCC  135 59.2   48 21.1 21.2 CTC 116 63.0  51 27.7 28.5 GCG  43 18.9   34 14.9 14.2 CTG  21 11.4  27 14.7 14.6 GCT   37 16.2   9340.8 41.3 CTT  44 23.9  59 32.1 31.6 ARG (R) AGA    0 0.0   54 45.0 43.8TTA   0 0.0   0 0.0 0.0 AGG    0 0.0   36 30.0 30.5 TTG   3 1.6  47 25.525.3 CGA    1 0.8    0 0.0 0.0 LYS (K) AAA  10 8.8  52 45.6 44.6 CGC  95 79.2    0 0.0 0.0 AAG 104 91.2  62 54.4 55.4 CGG    1 0.8    0 0.00.0 MET (M) ATG  45 100  45 100 100.0 CGT   23 19.2   30 25.0 25.7PHE (F) TTC  33 47.8  41 59.4 58.6 ASN (N) AAC   75 89.3   51 60.7 62.6TTT  36 52.2  28 40.6 41.4 AAT    9 10.7   33 39.3 37.4 PRO (P) CCA   87.2  33 29.7 29.6 ASP (D) GAC   86 72.3   52 43.7 42.5 CCC  47 42.3  1614.4 14.6 GAT   33 27.7   67 56.3 57.5 CCG  35 31.5  20 18.0 18.4CYS (C) TGC   41 100.0   20 48.8 49.2 CCT  21 18.9  42 37.8 37.3 TGT   0 0.0   21 51.2 50.8 SER (S) AGC  40 26.5  28 18.5 17.9 END TAA    1100.0    0 0.0 0.0 AGT   7 4.6  24 15.9 15.8 TAG    0 0.0    0 0.0 0.0TCA   2 1.3  31 20.5 20.4 TGA    0 0.0    1 100.0 100.0 TCC  55 36.4  2818.5 18.7 GLN (Q) CAA    8 13.6   30 50.8 50.0 TCG  33 21.9   0 0.0 0.0CAG   51 86.4   29 49.2 50.0 TCT  14 9.3  40 26.5 27.2 GLU (E) GAA   3324.8   58 43.6 43.6 THR (T) ACA   8 8.1  28 28.3 26.3 16 GAG  100 75.2  75 56.4 56.4 ACC  58 58.6  24 24.2 26.9 GLY (G) GGA   11 7.2   55 36.236.4 ACG  26 26.3  16 16.2 16.9 GGC  102 67.1   25 16.4 16.2 ACT   7 7.1 31 31.3 30.0 GGG    3 2.0   23 15.1 15.2 TRP (IV) TGG  22 100  22 100100.0 GGT   36 23.7   49 32.2 32.1 TYR (Y) TAC  51 91.1  32 57.1 59.4HIS (H) CAC   29 76.3   19 50.0 49.6 TAT   5 8.9  24 42.9 40.6 CAT    923.7   19 50.0 50.4 VAL (V) GTA   1 0.8   0 0.0 0.0 ILE (I) ATA    0 0.0  22 21.2 21.1 GTC  85 65.4  34 26.2 27.0 ATC   67 64.4   44 42.3 42.7GTG  30 23.1  42 32.3 31.7 ATT   37 35.6   38 36.5 36.2 GTT  14 10.8  5441.5 41.3 Totals 1079 1079 Totals 981 981

TABLE 5 PUFA chimeric OrfC codon compositions Plnt Plnt Plnt Plnt PlntPlnt Amino Original Original Opt Opt Opt Amino Original Original Opt OptOpt Acid Codon Gene # Gene % Gene # Gene % Recm'd Acid Codon Gene #Gene % Gene # Gene % Recm'd ALA (A) GCA  18 14.0  30 23.3 23.3 LEU (L)CTA   2 1.6   0 0.0 0.0 GCC  84 65.1  28 21.7 21.2 CTC  78 63.9  34 27.928.5 GCG  14 10.9  19 14.7 14.2 CTG   18 14.8  18 14.8 14.6 GCT  13 10.1 52 40.3 41.3 CTT  16 13.1  39 32.0 31.6 ARG (R) AGA   1 1.3  33 44.043.8 TTA   1 0.8   0 0.0 0.0 AGG   1 1.3  23 30.7 30.5 TTG   7 5.7  3125.4 25.3 CGA   6 8.0   0 0.0 0.0 LYS (K) AAA  15 16.1  42 45.2 44.6 CGC 53 70.7   0 0.0 0.0 AAG  78 83.9  51 54.8 55.4 CGG   3 4.0   0 0.0 0.0MET (M) ATG  48 100  48 100 100.0 CGT  11 14.7  19 25.3 25.7 PHE (F) TTC 40 58.8  40 58.8 58.6 ASN (N) AAC  63 90.0  43 61.4 62.6 TTT  28 41.2 28 41.2 41.4 AAT   7 10.0  27 38.6 37.4 PRO (P) CCA  10 11.2  27 30.329.6 ASP (D) GAC  70 76.9  40 44.0 42.5 CCC  35 39.3  13 14.6 14.6 GAT 21 23.1  51 56.0 57.5 CCG  26 29.2  16 18.0 18.4 CYS (C) TGC  26 81.3 16 50.0 49.2 CCT  18 20.2  33 37.1 37.3 TGT   6 18.8  16 50.0 50.8SER (S) AGC  16 19.0  13 15.5 17.9 END TAA   1 100.0   0 0.0 0.0 AGT   33.6  14 16.7 15.8 TAG   0 0.0   0 0.0 0.0 TCA   9 10.7  18 21.4 20.4 TGA  0 0.0   1 100.0 100.0 TCC  28 33.3  16 19.0 18.7 GLN (Q) CAA  11 24.4 25 55.6 50.0 TCG  21 25.0   0 0.0 0.0 CAG  34 75.6  20 44.4 50.0 TCT  7 8.3  23 27.4 27.2 GLU (E) GAA  17 19.1  40 44.9 43.6 THR (T) ACA   46.2  17 26.2 26.3 16 GAG  72 80.9  49 55.1 56.4 ACC  41 63.1  17 26.226.9 GLY (G) GGA  21 17.9  43 36.8 36.4 ACG   8 12.3  11 16.9 16.9 GGC 78 66.7  18 15.4 16.2 ACT  12 18.5  20 30.8 30.0 GGG   7 6.0  18 15.415.2 TRP (IV) TGG  18 100  18 100 100.0 GGT  11 9.4  38 32.5 32.1TYR (Y) TAC  41 87.2  28 59.6 59.4 HIS (H) CAC  24 85.7  14 50.0 49.6TAT   6 12.8  19 40.4 40.6 CAT   4 14.3  14 50.0 50.4 VAL (V) GTA   65.3   0 0.0 0.0 ILE (I) ATA   0 0.0  15 21.7 21.1 GTC  62 54.4  31 27.227.0 ATC  48 69.6  30 43.5 42.7 GTG  24 21.1  37 32.5 31.7 ATT  21 30.4 24 34.8 36.2 GTT  22 19.3  46 40.4 41.3 Totals 746 746 Totals 748 748

TABLE 6 Acyl-CoA synthetase codon compositions Plnt Plnt Plnt Plnt PlntPlnt Amino Original Original Opt Opt Opt Amino Original Original Opt OptOpt Acid Codon Gene # Gene % Gene # Gene % Recm'd Acid Codon Gene #Gene % Gene # Gene % Recm'd ALA (A) GCA   2 2.3  21 24.7 23.3 LEU (L)CTA   0 0.0   0 0.0 0.0 GCC  59 68.6  18 21.2 21.2 CTC  35 63.6  15 27.328.5 GCG  11 12.8  12 14.1 14.2 CTG   6 10.9   9 16.4 14.6 GCT  14 16.3 34 40.0 41.3 CTT  13 23.6  17 30.9 31.6 ARG (R) AGA   0 0.0  14 43.843.8 TTA   0 0.0   0 0.0 0.0 AGG   3 9.4  10 31.3 30.5 TTG   1 1.8  1425.5 25.3 CGA   0 0.0   0 0.0 0.0 LYS (K) AAA   2 4.1  22 44.9 44.6 CGC 25 78.1   0 0.0 0.0 AAG  47 95.9  27 55.1 55.4 CGG   0 0.0   0 0.0 0.0MET (M) ATG  21 100  21 100 100.0 CGT   4 12.5   8 25.0 25.7 PHE (F) TTC 16 51.6  18 58.1 58.6 ASN (N) AAC  22 95.7  14 60.9 62.6 TTT  15 48.4 13 41.9 41.4 AAT   1 4.3   9 39.1 37.4 PRO (P) CCA   0 0.0  11 30.629.6 ASP (D) GAC  38 74.5  22 43.1 42.5 CCC  20 55.6   5 13.9 14.6 GAT 13 25.5  29 56.9 57.5 CCG   9 25.0   7 19.4 18.4 CYS (C) TGC  11 91.7  6 50.0 49.2 CCT   7 19.4  13 36.1 37.3 TGT   1 8.3   6 50.0 50.8SER (S) AGC   7 17.5   7 17.5 17.9 END TAA   1 100.0   0 0.0 0.0 AGT   410.0   6 15.0 15.8 TAG   0 0.0   0 0.0 0.0 TCA   1 2.5   8 20.0 20.4 TGA  0 0.0   1 100.0 100.0 TCC  19 47.5   8 20.0 18.7 GLN (Q) CAA   3 18.8  8 50.0 50.0 TCG   7 17.5   0 0.0 0.0 CAG  13 81.3   8 50.0 50.0 TCT  2 5.0  11 27.5 27.2 GLU (E) GAA  11 17.7  27 43.5 43.6 THR (T) ACA   12.0  13 25.5 26.3 16 GAG  51 82.3  35 56.5 56.4 ACC  27 52.9  14 27.526.9 GLY (G) GGA   5 7.4  25 36.8 36.4 ACG  19 37.3   9 17.6 16.9 GGC 49 72.1  11 16.2 16.2 ACT   4 7.8  15 29.4 30.0 GGG   0 0.0  10 14.715.2 TRP (IV) TGG  10 100  10 100 100.0 GGT  14 20.6  22 32.4 32.1TYR (Y) TAC  18 85.7  12 57.1 59.4 HIS (H) CAC  10 83.3   6 50.0 49.6TAT   3 14.3   9 42.9 40.6 CAT   2 16.7   6 50.0 50.4 VAL (V) GTA   00.0   0 0.0 0.0 ILE (I) ATA   0 0.0  10 21.3 21.1 GTC  34 58.6  16 27.627.0 ATC  27 57.4  20 42.6 42.7 GTG   9 15.5  19 32.8 31.7 ATT  20 42.6 17 36.2 36.2 GTT  15 25.9  23 39.7 41.3 Totals 410 409 Totals 372 372

TABLE 7 Phosphopantetheinyl transferase HetI codon compositions PlntPlnt Plnt Plnt Plnt Plnt Amino Original Original Opt Opt Opt AminoOriginal Original Opt Opt Opt Acid Codon Gene # Gene % Gene # Gene %Recm'd Acid Codon Gene # Gene % Gene # Gene % Recm'd ALA (A) GCA   420.0   5 25.0 23.3 LEU (L) CTA   6 17.1   0 0.0 0.0 GCC   6 30.0   420.0 21.2 CTC   4 11.4  10 28.6 28.5 GCG   2 10.0   3 15.0 14.2 CTG   00.0   5 14.3 14.6 GCT   8 40.0   8 40.0 41.3 CTT   3 8.6  11 31.4 31.6ARG (R) AGA   1 6.3   6 37.5 43.8 TTA  14 40.0   0 0.0 0.0 AGG   1 6.3  5 31.3 30.5 TTG   8 22.9   9 25.7 25.3 CGA   2 12.5   0 0.0 0.0LYS (K) AAA  10 90.9   5 45.5 44.6 CGC   6 37.5   0 0.0 0.0 AAG   1 9.1  6 54.5 55.4 CGG   1 6.3   0 0.0 0.0 MET (M) ATG   1 100   1 100 100.0CGT   5 31.3   5 31.3 25.7 PHE (F) TTC   3 25.0   6 50.0 58.6 ASN (N)AAC   3 50.0   4 66.7 62.6 TTT   9 75.0   6 50.0 41.4 AAT   3 50.0   233.3 37.4 PRO (P) CCA   9 56.3   5 31.3 29.6 ASP (D) GAC   3 25.0   541.7 42.5 CCC   6 37.5   2 12.5 14.6 GAT   9 75.0   7 58.3 57.5 CCG   16.3   3 18.8 18.4 CYS (C) TGC   0 0.0   1 33.3 49.2 CCT   0 0.0   6 37.537.3 TGT   3 100.0   2 66.7 50.8 SER (S) AGC   0 0.0   2 15.4 17.9 ENDTAA   0 0.0   0 0.0 0.0 AGT   4 30.8   2 15.4 15.8 TAG   0 0.0   0 0.00.0 TCA   3 23.1   3 23.1 20.4 TGA   1 100.0   1 100.0 100.0 TCC   323.1   2 15.4 18.7 GLN (Q) CAA   5 45.5   5 45.5 50.0 TCG   1 7.7   00.0 0.0 CAG   6 54.5   6 54.5 50.0 TCT   2 15.4   4 30.8 27.2 GLU (E)GAA  13 72.2   8 44.4 43.6 THR (T) ACA   3 27.3   3 27.3 26.3 16 GAG   527.8  10 55.6 56.4 ACC   2 18.2   3 27.3 26.9 GLY (G) GGA   0 0.0   535.7 36.4 ACG   2 18.2   2 18.2 16.9 GGC   5 35.7   2 14.3 16.2 ACT   436.4   3 27.3 30.0 GGG   2 14.3   2 14.3 15.2 TRP (IV) TGG   6 100   6100 100.0 GGT   7 50.0   5 35.7 32.1 TYR (Y) TAC   2 22.2   5 55.6 59.4HIS (H) CAC   1 20.0   3 60.0 49.6 TAT   7 77.8   4 44.4 40.6 CAT   480.0   2 40.0 50.4 VAL (V) GTA   0 0.0   0 0.0 0.0 ILE (I) ATA   2 20.0  3 30.0 21.1 GTC   1 12.5   2 25.0 27.0 ATC   4 40.0   4 40.0 42.7 GTG  3 37.5   3 37.5 31.7 ATT   4 40.0   3 30.0 36.2 GTT   4 50.0   3 37.541.3 Totals 116 116 Totals 122 122

After the codon optimization of the coding region sequences werecompleted, additional nucleotide sequences were added to the optimizedcoding region sequence. Restriction sites for the facilitation ofcloning, a Kozak sequence and additional stop codons were added to theplant optimized coding sequence. In addition, a second series of PUFAsynthase OrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoAsynthetase and phosphopantetheinyl transferase HetI coding sequenceswere designed, which contained a chloroplast targeting sequence from theArabidopsis thaliana Ribulose Bisphosphate Carboxylase small chain 1A(GenBank ID: NM_202369.2). This sequence, SEQ ID NO: 28, was added tothe previously described coding sequences for PUFA synthase OrfA, PUFAsynthase OrfB, PUFA synthase chimeric OrfC and phosphopantetheinyltransferase HetI. The initial Methionine from SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8 and SEQ ID NO: 10 was removed and replaced with thechloroplast targeting sequence. The sequences that contain thechloroplast targeting sequence are identified as version 4 (v4)throughout the specification.

A second chloroplast transit peptide was added to the PUFA synthaseOrfA, PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoAsynthetase and phosphopantetheinyl transferase HetI coding sequences.These coding sequences were designed to contain a chloroplast targetingsequence from acyl-ACP-thioesterase (GenBank ID: X73849.1). Thissequence, SEQ ID NO: 29, was added to the previously described codingsequences for PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthasechimeric OrfC and phosphopantetheinyl transferase HetI. The initialMethionine from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:10 was removed and replaced with the chloroplast targeting sequence. Thesequences that contain the chloroplast targeting sequence are identifiedas version 5 (v5) throughout the specification.

An alternative version of the acyl-CoA synthetase gene fromSchizochytrium sp. was created by modifying the native gene sequence toremove superfluous open reading frames. This version was labeled as“SzACS-2 v4” and listed as SEQ ID NO: 30. The resulting gene is used toreplace the acyl-CoA synthetase expression gene sequence, describedabove as “SzACS-2 v3.”

Once a plant-optimized DNA sequence has been designed on paper or insilico, actual DNA molecules can be synthesized in the laboratory tocorrespond in sequence precisely to the designed sequence. Suchsynthetic DNA molecules can be cloned and otherwise manipulated exactlyas if they were derived from natural or native sources. Synthesis of DNAfragments comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ IDNO: 9 and SEQ ID NO: 10 containing the additional sequences describedabove were performed by commercial suppliers (Geneart Ag, Regensburg,Germany). The synthetic DNA was then cloned into expression vectors andtransformed into Agrobacterium and soybean as described in Examples 2and 3.

Example 2 Plasmid Construction for pDAB7362

The pDAB7362 binary plasmid (FIG. 2; SEQ ID NO:11) was constructed usinga multi-site Gateway L-R recombination reaction. pDAB7362 contains threePUFA synthase PTUs (which express the PUFA synthase OrfA, PUFA synthaseOrfB, PUFA synthase chimeric OrfC genes described above), one acyl-CoAsynthetase PTU, one phosphopantetheinyl transferase HetI PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains a truncated Phaseolus vulgarisphytohemagglutinin-L gene promoter (PvDlec2 promoter v2; GenBankAccession Number X06336), Arabidopsis thaliana AT2S3 gene 5′untranslated region (2S 5′ UTR; GenBank Accession Number NM 118850),Schizochytrium sp. Polyunsaturated Fatty Acid synthase Open ReadingFrame A (SzPUFA OrfA v3) and Arabidopsis thaliana 2S albumin gene 3′untranslated region terminator (At2S SSP terminator v1; GenBankAccession Number M22035). The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, Schizochytrium sp. Polyunsaturated FattyAcid synthase Open Reading Frame B (SzPUFA OrfB v3) and At2S SSPterminator v1. The third PUFA synthase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, Schizochytrium and Thraustochytrium sp. PolyunsaturatedFatty Acid synthase Open Reading Frame C (hSzThPUFA OrfC v3) and At2SSSP terminator v1. The acyl-CoA synthetase PTU contains the PvDlec2promoter v2, 2S 5′ UTR, Schizochytrium sp. acyl-CoA synthetase (SzACS-2v3) and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvDlec2 promoter v2, 2S 5′ UTR, Nostoc sp. 4′phosphopantetheinyl transferase HetI (No HetI v3) and At2S SSPterminator v1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB7339 and pDAB7333 wererecombined to form pDAB7362. Specifically, the five PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, SzACS-2 v3,NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: Cassava vein Mosaic Virus Promoter (CsVMV promoter v2;Verdaguer et al., Plant Molecular Biology 31:1129-1139; 1996),phosphinothricin acetyl transferase (PAT v5; Wohlleben et al., Gene70:25-37; 1988) and Agrobacterium tumefaciens ORF1 3′ untranslatedregion (AtuORF1 3′ UTR v4; Huang et al., J. Bacteriol. 172:1814-1822;1990), in addition to other regulatory elements such as Overdrive (Toroet al., PNAS 85(22): 8558-8562; 1988) and T-strand border sequences(T-DNA Border A and T-DNA Border B; Gardner et al., Science 231:725-727;1986 and International Publication No. WO 2001/025459 A1). Recombinantplasmids containing the five PTUs were then isolated and tested forincorporation of the five PTUs with restriction enzyme digestion and DNAsequencing.

Example 2.1 Construction of Additional Plasmids that use the PvDlec2Promoter to Drive Expression

Additional constructs were designed and built that use the PvDlec2promoter to drive expression of the PUFA synthase OrfA, PUFA synthaseOrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase, and 4′phosphopantetheinyl transferase HetI transgenes. Various alterations tothese constructs have been made to increase expression levels. Thesechanges include the use of non-codon optimized gene sequences,incorporation of chloroplast transit peptides, and removal of theacyl-CoA synthetase PTU.

The newly constructed plasmids are used to stably transform soybeanplants. Transgenic soybean plants are isolated and molecularlycharacterized. The use of these alternative constructs result in soybeanplants that contain greater amounts of DHA and LC-PUFAs. The resultingLC-PUFA accumulation is determined and soybean plants that produce 0.01%to 15% DHA or 0.01% to 15% LC-PUFA are identified.

Example 2.2 Construction of pDAB7361

pDAB7361 is a binary plasmid that was constructed to contain a native,non-codon optimized version of SzPUFA OrfA v2, the remaining genesequences are codon optimized (SzPUFA OrfB v3, hSzThPUFA OrfC v3,SzACS-2 v3, and NoHetI v3). The pDAB7361 plasmid (FIG. 3; SEQ ID NO:31)was constructed using a multi-site Gateway L-R recombination reaction.pDAB7361 contains three PUFA synthase PTUs, one acyl-CoA synthetase PTU,one phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v2 and At2S SSP terminatorv1. The second PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, SzPUFA OrfB v3 and At2S SSP terminator v1. The third PUFA synthasePTU contains the PvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfC v3 andAt2S SSP terminator v1. The acyl-CoA synthetase PTU contains the PvDlec2promoter v2, 2S 5′ UTR, SzACS-2 v3 gene and At2S SSP terminator v1. Thephosphopantetheinyl transferase PTU contains the PvDlec2 promoter v2, 2S5′ UTR, NoHetI v3 and At2S SSP terminator v1.

Plasmids pDAB7355, pDAB7335, pDAB7336, pDAB7339 and pDAB7333 wererecombined to form pDAB7361. Specifically, the five PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v2, SzPUFA OrfB v3, hSzThPUFA OrfC v3, SzACS-2 v3NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the six PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Example 2.3 Construction of DAB7363

pDAB7363 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA v4, SzPUFA OrfB v4, hSzThPUFAOrfC v4, and NoHetI v4 all of which contain the Ribulose BisphosphateCarboxylase small chain 1A (labeled as SSU-TP v1) that is fused to theamino terminus of the coding sequence. In addition this plasmid containsa rebuilt, codon optimized version of SzACS-2 v3. The pDAB7363 plasmid(FIG. 4; SEQ ID NO:32) was constructed using a multi-site Gateway L-Rrecombination reaction. pDAB7363 contains three PUFA synthase PTUs, oneacyl-CoA synthetase PTU, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v4and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v4 and At2S SSP terminatorv1. The third PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, hSzThPUFA OrfC v4 and At2S SSP terminator v1. The acyl-CoAsynthetase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzACS-2 v3gene and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvDlec2 promoter v2, 2S 5′ UTR, NoHetI v4 and At2S SSPterminator v1.

Plasmids pDAB7340, pDAB7341, pDAB7342, pDAB7344 and pDAB7333 wererecombined to form pDAB7363. Specifically, the five PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v4, SzPUFA OrfB v4, hSzThPUFA OrfC v4, SzACS-2 v3,NoHetI v4. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the six PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Example 2.4 Construction of pDAB7365

pDAB7365 is a binary plasmid that was constructed to contain native,non-codon optimized versions of SzPUFA OrfA v2, SzPUFA OrfB v2,hSzThPUFA OrfC v2, SzACS-2 v2, and NoHetI v2. The pDAB7365 plasmid (FIG.5; SEQ ID NO:33) was constructed using a multi-site Gateway L-Rrecombination reaction. pDAB7365 contains three PUFA synthase PTUs, oneacyl-CoA synthetase PTU, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v2and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v2 and At2S SSP terminatorv1. The third PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, SzPUFA OrfC v2 and At2S SSP terminator v1. The acyl-CoA synthetasePTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzACS-2 v2 gene andAt2S SSP terminator v1. The phosphopantetheinyl transferase PTU containsthe PvDlec2 promoter v2, 2S 5′ UTR, NoHetI v2 and At2S SSP terminatorv1.

Plasmids pDAB7355, pDAB7356, pDAB7357, pDAB7360 and pDAB7333 wererecombined to form pDAB7365. Specifically, the five PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v2, SzPUFA OrfB v2, SzPUFA OrfC v2, SzACS-2 v2,NoHetI v2. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the five PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Example 2.5 Construction of pDAB7368

pDAB7368 is a binary plasmid that was constructed to contain native,non-codon optimized versions of SzPUFA OrfA v2, SzPUFA OrfB v2,hSzThPUFA OrfC v2, and NoHetI v2. This construct does not contain theSzACS-2 coding sequence. The pDAB7368 plasmid (FIG. 6; SEQ ID NO:34) wasconstructed using a multi-site Gateway L-R recombination reaction.pDAB7368 contains three PUFA synthase PTUs, one acyl-CoA synthetase PTU,one phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v2 and At2S SSP terminatorv1. The second PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, SzPUFA OrfB v2 and At2S SSP terminator v1. The third PUFA synthasePTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfC v2 and At2SSSP terminator v1. The phosphopantetheinyl transferase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, NoHetI v2 and At2S SSP terminator v1.

Plasmids pDAB7355, pDAB7356, pDAB7357, pDAB7359 and pDAB7333 wererecombined to form pDAB7368. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v2, SzPUFA OrfB v2, SzPUFA OrfC v2, NoHetI v2.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 2.6 Construction of pDAB7369

pDAB7369 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFAOrfC v3, and NoHetI v3 this construct does not contain the SzACS-2coding sequence PTU. The pDAB7369 plasmid (FIG. 7; SEQ ID NO:35) wasconstructed using a multi-site Gateway L-R recombination reaction.pDAB7369 contains three PUFA synthase PTUs, one acyl-CoA synthetase PTU,one phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3 and At2S SSP terminatorv1. The second PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, SzPUFA OrfB v3 and At2S SSP terminator v1. The third PUFA synthasePTU contains the PvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfC v3 andAt2S SSP terminator v1. The phosphopantetheinyl transferase PTU containsthe PvDlec2 promoter v2, 2S 5′ UTR, NoHetI v3 and At2S SSP terminatorv1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB7338 and pDAB7333 wererecombined to form pDAB7369. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 2.7 Construction of pDAB7370

pDAB7370 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA v4, SzPUFA OrfB v4, hSzThPUFAOrfC v4, and NoHetI v4 that contain the Ribulose BisphosphateCarboxylase small chain lA (labeled as SSU-TP v1), which is fused to theamino terminus of the coding sequence. This construct does not containthe SzACS-2 coding sequence PTU. The pDAB7370 plasmid (FIG. 8; SEQ IDNO: 36) was constructed using a multi-site Gateway L-R recombinationreaction. pDAB7370 contains three PUFA synthase PTUs, one acyl-CoAsynthetase PTU, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v4and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v4 and At2S SSP terminatorv1. The third PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, hSzThPUFA OrfC v4 and At2S SSP terminator v1. Thephosphopantetheinyl transferase PTU contains the PvDlec2 promoter v2, 2S5′ UTR, NoHetI v4 and At2S SSP terminator v1.

Plasmids pDAB7340, pDAB7341, pDAB7342, pDAB7343 and pDAB7333 wererecombined to form pDAB7370. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v4, SzPUFA OrfB v4, hSzThPUFA OrfC v4, NoHetI v4.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 2.8 Construction of pDAB100518

pDAB100518 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA v5, SzPUFA OrfB v5, hSzThPUFAOrfC v5, and NoHetI v5 that contain the chloroplast transit peptide fromacyl-ACP-thioesterase (labeled as Thioesterase Transit Peptide), whichis fused to the amino terminus of the coding sequence. In addition, theplasmid contains a SzACS-2 v3 coding sequence PTU, which does notpossess a chloroplast transit peptide. The pDAB100518 plasmid (FIG. 9;SEQ ID NO:37) was constructed using a multi-site Gateway L-Rrecombination reaction. pDAB100518 contains three PUFA synthase PTUs,one acyl-CoA synthetase PTU, one phosphopantetheinyl transferase PTU anda phosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v5and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v5 and At2S SSP terminatorv1. The third PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, hSzThPUFA OrfC v5 and At2S SSP terminator v1. The acyl-CoAsynthetase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzACS-2 v3gene and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvDlec2 promoter v2, 2S 5′ UTR, NoHetI v5 and At2S SSPterminator v1.

Plasmids pDAB100517, pDAB100514, pDAB100511, pDAB100515 and pDAB7333were recombined to form pDAB100518. Specifically, the five PTUsdescribed above were placed in a head-to-tail orientation within theT-strand DNA border regions of the plant transformation binary pDAB7333.The order of the genes is: SzPUFA OrfA v5, SzPUFA OrfB v5, hSzThPUFAOrfC v5, SzACS-2 v3, NoHetI v5. pDAB7333 also contains thephosphinothricin acetyl transferase PTU: CsVMV promoter v2, PAT v5,AtuORF1 3′ UTR v4 in addition to other regulatory elements such asOverdrive and T-strand border sequences (T-DNA Border A and T-DNA BorderB). Recombinant plasmids containing the six PTUs were then isolated andtested for incorporation of the six PTUs with restriction enzymedigestion and DNA sequencing.

Example 2.9 Construction of pDAB101476

pDAB101476 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFAOrfC v3, and NoHetI v3. The SzACS-2 v2 gene sequence is the native,non-codon optimized version. The pDAB101476 plasmid (FIG. 10; SEQ ID NO:38) was constructed using a multi-site Gateway L-R recombinationreaction. pDAB101476 contains three PUFA synthase PTUs, one acyl-CoAsynthetase PTU, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v3 and At2S SSP terminatorv1. The third PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1. The acyl-CoAsynthetase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzACS-2 v2gene and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvDlec2 promoter v2, 2S 5′ UTR, NoHetI v3 and At2S SSPterminator v1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB101471 and pDAB7333 wererecombined to form pDAB101476. Specifically, the five PTUs describedabove were placed in a head-to-tail orientation within the T-strand DNAborder regions of the plant transformation binary pDAB7333. The order ofthe genes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, SzACS-2v2, NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5. AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the six PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Example 2.10 Construction of pDAB101477

pDAB101477 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFAOrfC v3, and NoHetI v3. The pDAB101477 plasmid (FIG. 11; SEQ ID NO:39)was constructed using a multi-site Gateway L-R recombination reaction.pDAB101477 contains three PUFA synthase PTUs, one acyl-CoA synthetasePTU, one phosphopantetheinyl transferase PTU and a phosphinothricinacetyl transferase PTU. Specifically, the first PUFA synthase PTUcontains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3 and At2S SSPterminator v1. The second PUFA synthase PTU contains the PvDlec2promoter v2, 2S 5′ UTR, SzPUFA OrfB v3 and At2S SSP terminator v1. Thethird PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR,hSzThPUFA OrfC v3 and At2S SSP terminator v1. The acyl-CoA synthetasePTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzACS-2 v4 gene andAt2S SSP terminator v1. The phosphopantetheinyl transferase PTU containsthe PvDlec2 promoter v2, 2S 5′ UTR, NoHetI v3 and At2S SSP terminatorv1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB101472 and pDAB7333 wererecombined to form pDAB101477. Specifically, the five PTUs describedabove were placed in a head-to-tail orientation within the T-strand DNAborder regions of the plant transformation binary pDAB7333. The order ofthe genes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, SzACS-2v4, NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the six PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Example 3 Soybean Transformation

Transgenic soybean (Glycine max) was generated throughAgrobacterium-mediated transformation of soybean cotyledonary nodeexplants. The disarmed Agrobacterium strain DA2552 (U.S. Appl. No.61/368,965, filed Jul. 29, 2010) carrying the binary vectors describedabove as pDAB7362 was used to initiate transformation.

Agrobacterium-mediated transformation was carried out using a modified ½cotyledonary node procedure of Zeng et al. (Zeng P., Vadnais D. A.,Zhang Z., Polacco J. C., (2004), Plant Cell Rep., 22(7): 478-482).Briefly, soybean seeds (cv. Maverick) were germinated on basal media andcotyledonary nodes were isolated and infected with Agrobacterium. Shootinitiation, shoot elongation, and rooting media were supplemented withcefotaxime, timentin and vancomycin for removal of Agrobacterium.Glufosinate selection was employed to inhibit the growth ofnon-transformed shoots. Selected shoots were transferred to rootingmedium for root development and then transferred to soil mix foracclimatization of plantlets.

Terminal leaflets of selected plantlets were treated topically (leafpaint technique) with glufosinate to screen for putative transformants.The screened plantlets were transferred to the greenhouse, allowed toacclimate and then leaf-painted with glufosinate to reconfirm tolerance.These putative transformed To plants were sampled and molecular analyseswas used to confirm the presence of PAT, and the PUFA synthase OrfA,PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase and4′ phosphopantetheinyl transferase HetI transgenes. To plants wereallowed to self-fertilize in the greenhouse to produce Ti seed.

A second soybean transformation method was used to produce additionaltransgenic soybean plants. The disarmed Agrobacterium strain DA2552(U.S. Provisional Patent App. No. 61/368,965) carrying the binary vectordescribed above as pDAB7362 was used to initiate transformation.

Agrobacterium-mediated transformation was carried out using a modifiedhalf-seed procedure of Paz et al., (M. Paz, J. Martinez, A. Kalvig, T.Fonger, and K. Wang (2005) Plant Cell Rep., 25: 206-213). Briefly,mature soybean seeds were sterilized overnight with chlorine gas, andimbibed with sterile H20 twenty hours before Agrobacterium-mediatedplant transformation. Seeds were cut in half by a longitudinal cut alongthe hilum to separate the seed and remove the seed coat. The embryonicaxis was excised and any axial shoots/buds were removed from thecotyledonary node. The resulting half seed explants were infected withAgrobacterium. Shoot initiation, shoot elongation, and rooting mediawere supplemented with cefotaxime, timentin and vancomycin for removalof Agrobacterium. Glufosinate selection was employed to inhibit thegrowth of non-transformed shoots. Selected shoots were transferred torooting medium for root development and then transferred to soil mix foracclimatization of plantlets.

Terminal leaflets of selected plantlets were treated topically (leafpaint technique) with glufosinate to screen for putative transformants.The screened plantlets were transferred to the greenhouse, allowed toacclimate and then leaf-painted with glufosinate to reconfirm tolerance.These putative transformed To plants were sampled and molecular analyseswas used to confirm the presence of PAT, and the PUFA synthase OrfA,PUFA synthase OrfB, PUFA synthase chimeric OrfC, acyl-CoA synthetase and4′ phosphopantetheinyl transferase HetI transgenes. Seven events wereidentified as containing the transgenes from pDAB7362. These To plantswere advanced for further analysis and allowed to self-fertilize in thegreenhouse to give rise to Ti seed.

Example 4 Molecular Analysis of Soybean Events

Transgene copy numbers of selected pDAB7362 soybean events werequantified using a comparative quantitative real time PCR (qPCR) method.Leaf tissue samples were taken from the top and bottom leaves of amature soybean plant, these samples were combined and the genomic DNAwas isolated. Genomic DNA was isolated using the BioSprint 96 DNA PlantKit and a BioSprint 96 magnetic particle automation platform (Qiagen,Valencia, Calif.) per manufacturer's instructions. Extracted genomic DNAwas diluted 1:5 with ddH20 for use as template in quantitative real timePCR reactions (qPCR).

qPCR Assays were designed to detect the SzPUFA OrfA v3, SzPUFA OrfB v3,hThSzPUFA OrfCv3, SzACS-2 v3, NoHetI v3, and PAT v5 transgenes inpDAB7362 soybean plants by using the Roche Assay Design Center(www.universalprobelibrary.com). The primers and probes used in theassays are described in Table 8. The presences of the target genes weredetected with fluorescein-amidite (FAM) labeled UPL probes (RocheDiagnostics, Indianapolis, Ind.). These assays were executed in duplexreactions with a soybean internal reference GMFL01-25-J19, GenBank:AK286292.1 (referenced as GMS116 in Table 8), which was labeled with theCyanine-5 (Cy-5) fluorescent dye.

TABLE 8 qPCR assay primers and probes Target Forward primerReverse Primer Probe SzPUFA SEQ ID NO: 12 SEQ ID NO: 13 UPL #18 OrfA v3cacaaccggtgttgatgatg Gagcttcacaaaggctctgc SzPUFA SEQ ID NO: 14SEQ ID NO: 15 UPL #97 Orf13 v3 gaatccttgcgtcatttggt CaatggactcacgcacaacthThSzPUFA SEQ ID NO: 16 SEQ ID NO: 17 UPL #26 OrfCv3ggattacctcaacattgctcct Tgtccatgcgcatatcctt SzACS2 v3 SEQ ID NO: 18SEQ ID NO: 19 UPL #54 agaaattgatggctgttggtg CtgccgtgctgagtttcttNoHetI v3 SEQ ID NO: 20 SEQ ID NO: 21 UPL #3 ccagaacacagaaggcgtttTcccaagtatccacccaagat PAT v5 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24acaagagtggattgatgatctagagaggt Ctttgatgcctatgtgacacgtaaacagtccagcgtaagcaata ccagccacaacacc GMS116 SEQ ID NO: 25 SEQ ID NO: 26SEQ ID NO: 27 gtaatatgggctcagaggaatggt atggagaagaacattggaattgcccatggcccggtacc atctggtc

Real-time PCR reactions were run on a LC48011 real-time PCR thermalcycler (Roche, Indianapolis, Ind.) using standard protocols. Data forthe SzPUFA OrfA v3, SzPUFA OrfB v3, hThSzPUFA OrfCv3, SzACS-2 v3, NoHetIv3, and PAT v5, FAM-labeled assays were collected using a 533 nmemission filter and a 483 nm excitation signal. Data for the GMS1.16Cy5-labeled reference assay was collected using a 660 nm filter and a618 nm excitation signal. Crossing point values (Cp values) and targetto reference ratios were calculated automatically using the LC480IIsoftware's “Advanced Relative Quantification” analysis workflow. Atarget-to-reference ratio for each sample was calculated using thestandard “delta-delta-Ct” method. Estimated copy number was determinedby normalizing sample target-reference ratios with the target-referenceratio of the soybean internal reference GMFL01-25-J19.

The estimated copy number of the PAT v5 selectable marker anddocosahexaenoic acid (DHA) transgenes (SzPUFA OrfA v3, SzPUFA OrfB v3,hThSzPUFA OrfC v3, SzACS-2 v3, and NoHetI v3) was determined in Tiplants from the seven pDAB7362 events. Plants from two events,7362[710]-71006 and 7362[710]-71010, did not contain either the PAT v5selectable marker or the DHA gene target sequences. Plants from theremaining events; 7362[710]-70903, 7362[710]-71005, 7362[710]-71008, and7362[710]-71009, contained the PAT v5 selectable marker and the five DHAtransgenes with copy numbers ranging from 1-10. Event 7362[708]-70801produced T1 plants with 0, 1 or 2 copies of the PAT v5 gene indicating asingle segregating locus and event 7362 [710]-71005 produced T1 plantswith PAT v5 copy numbers between 0 and 4 suggesting segregation of twounlinked loci.

Example 5 Lipid Analysis of T₁ Cotyledons of Transgenic Soybean Plants

To avoid destructive analysis of limited quantities of T₁ seeds, fattyacid methyl ester (FAMES) analysis was performed on post-germinationgreen cotyledons of T₁ plants. Methods for the purification and analysisof FAMEs have been described (e.g., Z. D. Nightingale et al. (1999),Purification of fatty acid methyl esters by high-performance liquidchromatography, J. Chromatogr. B. Biomed. Sci. Appl. 732(2):495-500; and“Gas chromatography and lipids: a practical guide” by W. W Christie,1989, The Oily Press). Characterization of the oil profile in the T₁cotyledons is indicative of the oil profile in dry T₁ seed (R. F. Wilsonand P. Kwanyuen (1986), Triacylglycerol synthesis and metabolism ingerminating soybean cotyledons, Biochimica et Biophysica Acta(BBA)—Lipids and Lipid Metabolism, 877(2):231-237).

Example 5.1 Validation of Post-Germination Detection of DHA in TiCotyledons via Analysis of Transgenic Canola

Validation and detection of Long Chain Poly Unsaturated Fatty Acids(LC-PUFA) in post-germination green cotyledons was performed withDHA-producing canola seed to assess if characterization of the oilprofile in the T₁ cotyledons is indicative of the presence of an oilprofile within the mature T₁ seed. Transgenic canola seed harboring thebinary plasmid, pDAB7362, were germinated at room temperature onwater-saturated paper towels, and harvested after 3 days at which pointthe tissue was lyophilized. The tissue was directly transmethylated andnot pre-extracted with hexane. The LC-PUFA content (% FAMEs by weight)was calculated and compared to the mature seed. The average DHA contentfrom the 30 canola emerged cotyledons was 0.71% (total LC-PUFA=0.97%)with an oil content of 53.0%. The average DHA content of 48 maturecanola seed prior to germination was 0.49% (total LC-PUFA=0.73%) with anoil content of 44.3%. This study demonstrates that LC-PUFAs can bedetected post-germination in emerged green cotyledons and that detectionof the LC-PUFAs in emerged green cotyledons indicates that LC-PUFA ispresent in the seed.

Example 5.2 Post-Germination Detection of DHA in T₁ Soybean Cotyledons

FAME analysis was performed on one excised green cotyledon per soybeanseedling sampled 3 to 5 days after planting. The plant material waslyophilized, homogenized using a steel ball and ball mill and defatted 3times with hexane. The pooled hexane fraction was evaporated and the dryresidue was weighed and reconstituted in heptane. A known amount of oilresidue was transmethylated with 0.25 M of freshly prepared sodiummethoxide (Sigma-Aldrich, St. Louis, Mo.) in methanol in the presence ofthe surrogate, triheptadecanoin (Nu-Chek Prep, Elysian, Minn.). Thereaction was conducted under mild heat (40° C.) and constant shaking andthe resulting FAMEs extracted with heptane. Completion of the reactionwas verified by recovery of the reacted heptadecanoate methyl-estersurrogate. The FAMEs extracts were analyzed by GC-FID using an Agilent6890 Gas Chromatograph (Agilent Technologies, Santa Clara, Calif.) and a15 m×0.25 mm×0.25 μm BPX 70 capillary column from SGE (Austin, Tex.).Each FAME peak was identified by its retention time and quantified bythe injection of a rapeseed oil reference mix from Matreya LLC (PleasantGap, Pa.). The calibration standard contained individually addedstandards of DHA, EPA and DPA(n-6) methyl esters from Nu-Chek. Dataanalysis was performed using ChemStation4 software (Agilent). Ticotyledons from two events contained DHA; pDAB7362[708]-70801.001 andpDAB7362[710]-71005.001 (Table 9).

Forty seeds from Event pDAB7362[708]-70801.001 were germinated andscreened for the presence of LC-PUFA in excised green cotyledon.Cotyledons from six of the forty seeds contained LC-PUFA in a range of0.78% to 1.58% (with a mean of 1.12%). DHA content ranged from 0.48% to0.93% (with a mean of 0.68%), and DP A (n-6) content ranged from 0.3% to0.65% (with a mean of 0.44%).

Thirty-nine seeds from Event pDAB7362[710]-71005.001 were germinated andscreened for the presence of LC-PUFA in excised green cotyledon.Cotyledons from thirty-seven of the thirty-nine seeds contained LC-PUFAin a range of 0.70% to 1 1.98% (with a mean of 3.91%). Of the totalLC-PUFA, DHA content ranged from 0.36% to 8.00% (with a mean of 2.24%),and DPA(n-6) content ranged from 0.34% to 3.98% (with a mean of 1.68%).

Identification of LC-PUFA was confirmed by evaluating specificfragmentation of standard PUFA methyl esters (Nu-Chek Prep, Elysian,Minn.) using a Pegasus III GC-TOF-MS (Leco, St. Joseph, Mich.) comparedto a negative control.

TABLE 9 LC-PUFA content by weight percentage of total fatty acids fromgerminated T₁ soybean seed cotyledons # of DHA # of total positive DHADPA(n-6) Total PUFA Event ID seedlings seedlings Mean Range Mean RangeMean Range pDAB7362[708]- 40 6 0.68% 0.48-0.93% 0.44%  0.3-0.65% 1.12% 0.78-1.58% 70801.001 pDAB7362[710]- 39 37 2.24% 0.36-8.00% 1.68%0.34-3.98% 3.91% 0.70-11.98% 71005.001 Williams 82 15 0   0% —   0% —  0% — control

Example 6 Lipid Analysis of Mature T₂ seed from Transgenic SoybeanEvents

T₁ plants from two events, 7362[708]-70801.001 and 7362[710]-71005.001,were grown to maturity in the greenhouse. Plants were selected thatcontained high levels of LC-PUFAs in the T₁ cotyledon and one or twocopies of PAT v5 and the accompanying five genes for DHA production.These plants were self-fertilized and the resulting T₂ seed harvested atmaturity. Single seeds were analyzed via FAMEs GC-FID to determine theLC-PUFA and DHA content in the T₂ soybean seed. Twelve whole matureseeds per plant were individually analyzed by crushing the seed with apress and homogenization using a steel ball and ball mill. The tissuewas defatted three times with hexane, the pooled hexane fractions wereevaporated to dryness and the residue weighed and reconstituted inheptane for FAME analysis performed as described in the previousexample.

Single T₂ seeds from a T₁ plant of event 7362[708]-70801.001 (describedas 7362[708]-70801.Sx.021 in FIG. 12) that possessed a single copy ofPAT v5 contained 0% to 0.73% DHA (0% to 1.19% total LC-PUFA). Single T₂seeds from two T₁ plants of event 7362[710]-71005.001 (described as7362[710]-71005.Sx.006 and 7362[710]-71005.Sx.0.35 in FIG. 12)possessing a single copy of PAT v5 contained 0% to 2.08% DHA (0% to3.56% total LC-PUFA). Single T₂ seeds from seven T₁ plants of event7362[710]-71005.001 (described as 7362[710]-71005.Sx.010,7362[710]-71005.Sx.012, 7362[710]-71005.Sx.013, 7362[710]-71005.Sx.016,7362[710]-71005.Sx.018, 7362[710]-71005.Sx.025, and 7362[710]-71005Sx.031 in FIG. 12) containing two copies of PAT v5 contained 0% to 2.84%DHA (0% to 4.77% total LC-PUFA). The mean DHA content of T2 seeds fromthe highest DHA-producing line (7362[710]-71005.5x.025) was 1.83% (3.11%total LC-PUFA). The DHA content of each T₂ seed from the individual T₁plants is shown in FIG. 12.

DHA comprised 60% of the total LC-PUFA content in those T₂ seeds thatcontained LC-PUFA. Only the two novel LC-PUFAs, DHA and DPA(n-6), weredetected in the T₂ soybean seeds. The fatty acids that are expected tobe found in soybean seeds were detected at normal levels, except thattotal CI 8 fatty acids were proportionally lower due to the presence ofLC-PUFAs. No other different fatty acids were detected in thesetransgenic soybean seeds other than DHA and DPA(n-6). The oil content(sum of the masses of the individual FAMEs divided by seed mass) of thetransgenic seeds and the number of seeds produced by the transgenic T₁lines was not significantly different from that of the nontransgenicWilliams 82 control cultivar grown in the greenhouse at the same timeunder the same conditions.

The complete FAMEs profiles of individual T₂ seeds from soybean events7362[708]-70801.001 and 7362[710]-71005.001 are shown in Table 10.

TABLE 10 FAMEs profiles of individual T₂ soybean seeds from two events7362[708]-70801.001 and 7362[710]-71005.001. Values are percentages ofthe total FAME content from the 10 to 12 T₂ soybean seeds. Total LC-PUFAis the sum of C22:5 (DPA n-6) and C22:6 (DHA) FAME percentage. C22:5(DPA C22:6 Total Event Name C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0C20:1 C22:0 C24:0 n-6) (DHA) LC-PUFA 7362[708]- 0.16 12.99 4.98 13.8458.09 7.88 0.36 0.16 0.22 0.11 0.46 0.73 1.19 70801.Sx.021 7362[710]-0.19 13.09 4.8 22.71 50.56 5.37 0.32 0.17 0.25 0.1 0.83 1.61 2.4471005.Sx.006 7362[710]- 0.15 12.88 5.56 16.25 55.06 6.44 0.38 0.17 0.180.11 1.19 1.63 2.82 71005.Sx.010 7362[710]- 0.18 13.41 4.15 13.67 56.637.09 0.35 0.16 0.21 0.11 1.58 2.46 4.04 71005.Sx.012 7362[710]- 0.2214.3 4.4 19.13 51.2 5.89 0.31 0.15 0.18 0.1 1.53 2.59 4.12 71005.Sx.0137362[710]- 0.21 14.3 4.16 15.41 55.84 6.24 0.3 0.15 0.17 0.08 1.23 1.913.15 71005.Sx.016 7362[710]- 0.17 13.88 4.65 15.3 55.14 6.26 0.35 0.160.19 0.08 1.5 2.32 3.82 71005.Sx.018 7362[710]- 0.17 13.29 4.74 15.0354.94 6.2 0.37 0.17 0.21 0.09 1.93 2.84 4.77 71005.Sx.025 7362[710]- 013.42 4.5 16.4 55.73 6.13 0.19 0.1 0.24 0.06 1.27 1.96 3.23 71005.Sx.0317362[710]- 0.16 13.13 3.96 18.39 53.15 7.18 0.14 0.11 0.21 0 1.49 2.083.56 71005.Sx.035 Williams 82 0.06 10.37 6.25 24.12 52.88 5.46 0.33 0.140.3 0.09 0 0 0 Control

Example 6.1 Lipid Analysis of Mature T₃ seed from two Transgenic SoybeanEvents

Two T₂ soybean plant events, 7362[708]-70801.001 and7362[710]-71005.001, were grown to maturity in the greenhouse. Multipleplants of each event were grown in the greenhouse, and were screened toidentify individual plants that produced high levels of LC-PUFAs in theT₂ cotyledon and contained a single, homozygous insertion of thetransgenes. Identified plants were self-fertilized and the resulting T₃seed was harvested when the seed reached maturity. Single mature T₃seeds were analyzed via FAMEs GC-FID to determine the DHA and LC-PUFAcontent in the T₃ soybean seed (FIG. 12a ). Twelve whole mature seedsper plant were individually analyzed by crushing the seed with a pressand homogenizing the crushed seed material using a steel ball and ballmill. The tissue was defatted three times with hexane, the pooled hexanefractions were evaporated to dryness and the residue weighed andreconstituted in heptane for FAME analysis performed as described in theprevious example. The DHA levels were determined from the T₃ seed andcompared to the T₂ DHA levels, which had been assayed previously (Table11).

TABLE 11 Average DHA content (%) from randomly chosen mature soybeanseed at the T2 and T3 generation from two events 7362[708]70801.001 and7362[710]-71005.001. T₂ seed T₃ seed Event Name n Mean Min Max n MeanMin Max pDAB7362[708] 9 0.22 0 0.73 90 0.27 0 0.93 70801.001-1-21pDAB7362[710] 12 1.79 0.82 2.59 45 2.11 0.79 3.91 71005.001-1-13pDAB7362[710] 12 1.58 0.79 2.32 48 2.00 1.05 3.54 71005.001-1-18pDAB7362[710] 12 1.83 0.99 2.84 39 2.02 0.99 4.24 71005.001-1-25pDAB7362[710] 12 0.59 0 2.08 72 0.74 0 3.10 71005.001-1-35 Williams 82 80 0 0 15 0 0 0 Control

As indicated in Table 11, the relative percentage of DHA in soybeanseeds remained constant or increased in subsequent generations ofsoybean (from the T₂ and T₃). Single T₃ seeds produced fromself-fertilization of a T₂ plant of event 7362[708]-70801.001 (this linewas molecularly characterized and found to possess a single hemizygouscopy of PAT) were assayed via the FAMEs analysis and the seeds weredetermined to contain from 0% to 0.93% DHA (0% to 1.37% total LC-PUFA).Comparatively, the T₂ seeds produced from event 7362[708]-70801.001 wereassayed via FAMEs analysis and the seeds were determined to contain from0% to 0.73% DHA. Single T₃ seeds from self-fertilization of a T₂ plantevent 7362[710]-71005.001-1-35 (this line was molecularly characterizedand found to possess a single hemizygous copy of PAT) were assayed viathe FAMEs analysis and the seeds were determined to contain 0% to 3.10%DHA (0% to 5.45% total LC-PUFA). Comparatively, the T₂ seeds producedfrom event 7362[710]-71005.001-1-35 were assayed via FAMEs analysis andthe seeds were determined to contain from 0% to 2.84% DHA. In addition,Single T₃ seeds produced from events 7362[710]-71005.001-1-13,7362[710]-71005.001-1-18, and 7362[710]-71005.001-1-25 (each event wasdetermined to contain a single, homozygous copy of PAT) contained 0.79%to 4.24% DHA (1.26% to 6.5% total LC-PUFA). Comparatively, the T2 seedsproduced from events 7362[710]-71005.001-1-13, 7362[710]-71005.001-1-18,and 7362[710]-71005.001-1-25 were assayed via FAMEs analysis and theseeds were determined to contain from 0.79% to 2.84% DHA. The transgenicevents were compared to the control plants, the yield per plant (numberof seed) and total oil content (%) was found to be similar to theWilliams 82 control in similar conditions as the transgenic lines.

For all lines tested, the percentage of DHA and LC-PUFA that wasproduced and measured in the soybean seed for the T₂ and T₃ generationswas either consistent or increased in levels from the T₂ generation tothe T₃ generation. These results indicate that the traits are heritable,and that the transmission of the traits to further generations does notresult in reduced DHA production.

Example 7 Western Blot Detection of PUFA Synthase Proteins in TransgenicSoybean Seed

PUFA synthase OrfA (encoded by SzPUFA OrfA v3 gene), PUFA synthase OrfB(encoded by SzPUFA OrfB v3 gene) PUFA synthase chimeric OrfC (encoded byhThSzPUFA OrfC v3 gene) and HetI (from Nostoc sp. PCC 7120, GenBank ID:P37695, GL20141367) were detected in mature transgenic seed samples byWestern blot analysis. Residual soybean T₂ seed cake samples wereretained after the hexane extraction for FAME analysis. The powderedseed cake was placed in a tube with a single 4.5 mm stainless steel balland extraction buffer (50 mM Tris, 10 mM EDTA, 2% SDS) was added. Thesample tubes were rocked gently for 30 minutes, centrifuged for 15minutes at 3,000 rcf and the supernatant was used for analysis. Theamount of total soluble protein in the seed extract was determined by660 nm Protein Assay (Thermo Fisher, Rockford, Ill.). Samples werenormalized to 1.25 mg/ml total soluble protein and prepared in LDSsample buffer (Invitrogen, Carlsbad, Calif.) with 50 mM DTT for anormalized load of 16.25 μg total soluble protein per lane. Samples wereelectrophoresed in 3%-8% Tris-acetate gels (Invitrogen, Carlsbad,Calif.) and transferred to nitrocellulose membranes for detection ofPUFA synthase OrfA, PUFA synthase OrfB, and PUFA synthase chimeric OrfC.Samples were electroporesed in 4%-12% Bis-Tris gels (Invitrogen,Carlsbad, Calif.) and transferred to nitrocellulose membranes fordetection of HetI.

Blots were incubated in blocking buffer then probed with antibodiesagainst the different PUFA synthase OrfA, PUFA synthase OrfB, PUFAsynthase chimeric OrfC, and HetI polypeptides. The rabbit anti-A2-A thatis directed against the A2 region of Schizochytrium PUFA Synthase OrfA(SzPUFS-A), the rabbit anti-B3-A that is directed against the B3 regionof Schizochytrium PUFA Synthase OrfB (SzPUFS-B), and the rabbitanti-HetI that is directed against the full length HetI polypeptide wereused. Region B3 includes the Enoyl Reductase (ER) domain of OrfB. Asthere is also a homologous ER domain in PUFA synthase chimeric OrfC,this antiserum recognizes both PUFA synthase OrfB and PUFA synthasechimeric OrfC on a western blot. An anti-rabbit fluorescent labeledsecondary antibody (Goat Anti-Rabbit AF 633 (Invitrogen, Carlsbad,Calif.)) was used for detection. Blots were visualized on a Typhoon TrioPlus fluorescent imager (GE Healthcare, New Brunswick N.J.).

SDS-PAGE western blots of proteins extracts from mature T2 seed fromevents 7362[708]-70801 and 7362[710]-71005 showed bands at theappropriate size when probed with PUFA synthase OrfA, PUFA synthaseOrfB, PUFA synthase chimeric OrfC, and HetI specific antisera (FIG. 13).The bands for PUFA synthase OrfA, PUFA synthase OrfB, and PUFA synthasechimeric OrfC could also be seen by direct staining with Coomassie Blue.

Example 8 Expression of the Algal PUFA Synthase Gene Suite UsingAlternative Promoters

The use of additional transcriptional regulatory elements to express thegene(s) encoding PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthasechimeric OrfC, acyl-CoA synthetase and 4′ phosphopantetheinyltransferase HetI proteins can further increase LC-PUFA and DHA contentwithin soybean seeds. Identification and use of transcriptionalregulatory elements that express earlier in development duringtriacylglycerol biosynthesis and deposition, and for extended periods oftime can increase the levels of LC-PUFA and DHA within soybean seed bypromoting transcription of a LC-PUFA and DHA biosynthetic genes atearlier stages of seed development (e.g., at 15 to 25 DAP) and thereforeextend the time of LC-PUFA and DHA production. Examples of suchtranscriptional regulatory regions include, but are not limited to, theLesquerella fendleri KCS (LfKCS3) promoter (U.S. Pat. No. 7,253,337) andthe FAE 1 promoter (U.S. Pat. No. 6,784,342) and the Brassica oleraceaAcyl Carrier Protein (BoACP) promoter (International Publ. No. WO1992/18634). In addition, other seed specific promoters such as thephaseolin promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200) canbe used to robustly drive expression of heterologous genes for extendedperiods of time during seed development to increase the levels ofLC-PUFA and DHA within soybean seed. Finally, strong constitutivepromoters such as the Cassava Vein Mosaic Virus promoter (CsVMV promoterv2) can be used to drive expression of the heterologous genes throughoutall stages of development, thereby increasing the levels of LC-PUFA andDHA within soybean seed and other plant tissues.

These promoters are used singularly or in combination to drive theexpression of the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthasechimeric OrfC, acyl-CoA synthetase and 4′ phosphopantetheinyltransferase HetI expression cassettes, which were previously describedin plasmid, pDAB7362. Methods to replace transcriptional regulatoryregions within a plasmid are well known within the art. As such, apolynucleotide fragment comprising the PvDlec2 promoter v2 is removedfrom pDAB7362 (or the preceding plasmids used to build pDAB7362) andreplaced with new promoter regions. The newly constructed plasmids areused to stably transform soybean plants. Transgenic soybean plants areisolated and molecularly characterized. The resulting LC-PUFAaccumulation is determined by analyzing the lipid profiles (FAMEs) usingmethods described herein, and soybean plants that produce 0.01% to 15%DHA by weight of total fatty acids, 0.01% to 10% DPA(n-6) by weight oftotal fatty acids, or 0.01% to 10% EPA by weight of total fatty acidsare identified.

Use of Promoters That Express Early in Seed Development Example 8.1Construction of pDAB9166

The pDAB9166 plasmid (FIG. 14; SEQ ID NO:40) was constructed using amulti-site Gateway L-R recombination reaction. pDAB9166 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the LfKCS3 promoter v1, SzPUFA OrfA v3 andAtuORF23 3′ UTR v1. The second PUFA synthase PTU contains the LfKCS3promoter v1, SzPUFA OrfB v3 and AtuOrf23 3′ UTR v1. The third PUFAsynthase PTU contains the Lf CS3 promoter v1, hSzThPUFA OrfC v3 andAtuORF23 3′ UTR v1. The phosphopantetheinyl transferase PTU contains theLfKCS3 promoter v1, NoHetI v3 and AtuORF23 3′ UTR v1.

Plasmids pDAB9161, pDAB9162, pDAB9163, pDAB101484 and pDAB7333 wererecombined to form pDAB9166. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 8.2 Construction of pDAB9167

The pDAB9167 plasmid (FIG. 15; SEQ ID NO:41) was constructed using amulti-site Gateway L-R recombination reaction. pDAB9167 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the LfKCS3 promoter v1, SzPUFA OrfA v3 andAtuORF23 3′ UTR v1. The second PUFA synthase PTU contains the BoACPpromoter v1, BoACP 5′ UTR v1, SzPUFA OrfB v3 and AtuOrf23 3′ UTR v1. Thethird PUFA synthase PTU contains the LfKCS3 promoter v1, hSzThPUFA OrfCv3 and AtuORF23 3′ UTR v1. The phosphopantetheinyl transferase PTUcontains the BoACP promoter v1, BoACP 5′ UTR v1, NoHetI v3 and AtuORF233′ UTR v1.

Plasmids pDAB9161, pDAB9165, pDAB9163, pDAB101485 and pDAB7333 wererecombined to form pDAB9167. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Plasmids Containing the Phaseolin Promoter Example 8.3 Construction ofpDAB7379

pDAB7379 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,and NoHetI. The SzACS-2 gene sequence is not included in this construct.The pDAB7379 plasmid (FIG. 16; SEQ ID NO:42) was constructed using amulti-site Gateway L-R recombination reaction.

pDAB7379 contains three PUFA synthase PTUs, one phosphopantetheinyltransferase PTU and a phosphinothricin acetyl transferase PTU.Specifically, the first PUFA synthase PTU contains the PvPhas Promoterv3, PvPhas 5′ UTR, SzPUFA OrfA v3 and AtuORF23 3′ UTR v1. The secondPUFA synthase PTU contains the PvPhas Promoter v3, PvPhas 5′ UTR, SzPUFAOrfB v3 and AtuORF23 3′ UTR v1. The third PUFA synthase PTU contains thePvPhas Promoter v3, PvPhas 5′ UTR, hSzThPUFA OrfC v3 and AtuORF23 3′ UTRv1. The phosphopantetheinyl transferase PTU contains the PvPhas Promoterv3, PvPhas 5′ UTR, NoHetI v3 and AtuORF23 3′ UTR v1.

Plasmids pDAB7371, pDAB7372, pDAB7373, pDAB7374 and pDAB7333 wererecombined to form pDAB7379. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 8.4 Construction of DAB7380

pDAB7380 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,and NoHetI. The SzACS-2 gene sequence is not contained in thisconstruct. The version of the phaseolin promoter used in this constructwas modified essentially as described in Bustos et al., 1989 (The PlantCell, Vol. 1; 839-853), wherein the 5′ portion of the promoter wastruncated and the phaseolin 5′ untranslated region was left intact. ThepDAB7380 plasmid (FIG. 17; SEQ ID NO:43) was constructed using amulti-site Gateway L-R recombination reaction.

pDAB7380 contains three PUFA synthase PTUs, one phosphopantetheinyltransferase PTU and a phosphinothricin acetyl transferase PTU.Specifically, the first PUFA synthase PTU contains the PvPhas Promoterv4, PvPhas 5′ UTR, SzPUFA OrfA v3 and AtuORF23 3′ UTR v1. The secondPUFA synthase PTU contains the PvPhas Promoter v4, PvPhas 5′ UTR, SzPUFAOrfB v3 and AtuORF23 3′ UTR v1. The third PUFA synthase PTU contains thePvPhas Promoter v4, PvPhas 5′ UTR, hSzThPUFA OrfC v3 and AtuORF23 3′ UTRv1. The phosphopantetheinyl transferase PTU contains the PvPhas Promoterv5, PvPhas 5′ UTR, NoHetI v3 and AtuORF23 3′ UTR v1.

Plasmids pDAB7375, pDAB7376, pDAB7377, pDAB7378 and pDAB7333 wererecombined to form pDAB7380. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 8.5 Construction of DAB9323

pDAB9323 is a binary plasmid that was constructed to contain native,non-codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFAOrfC, SzACS-2, and NoHetI. The pDAB9323 plasmid (FIG. 18; SEQ ID NO:44)was constructed using a multi-site Gateway L-R recombination reaction.

pDAB9323 contains three PUFA synthase PTUs, one acyl-CoA synthetase PTU,one phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains thePvPhas Promoter v3, PvPhas 5′ UTR, SzPUFA OrfA v2, PvPhas 3′ UTR v1 andPvPhas 3′ MAR v2 (unannotated on the plasmid map). The second PUFAsynthase PTU contains the PvPhas Promoter v3, PvPhas 5′ UTR, SzPUFA OrfBv2, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2 (unannotated on the plasmidmap). The third PUFA synthase PTU contains the PvPhas Promoter v3,PvPhas 5′ UTR, SzPUFA OrfC v2, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map). The acyl-CoA synthetase PTU containsthe PvPhas Promoter v3, PvPhas 5′ UTR, SzACS-2 v2 gene, PvPhas 3′ UTR v1and PvPhas 3′ MAR v2 (unannotated on the plasmid map). Thephosphopantetheinyl transferase PTU contains the PvPhas Promoter v3,PvPhas 5′ UTR, NoHetI v2, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map).

Plasmids pDAB9307, pDAB9311, pDAB9315, pDAB9322 and pDAB7333 wererecombined to form pDAB9323. Specifically, the five PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v2, SzPUFA OrfB v2, SzPUFA OrfC v2, NoHetI v2.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thesix PTUs were then isolated and tested for incorporation of the six PTUswith restriction enzyme digestion and DNA sequencing.

Example 8.6 Construction of pDAB9330

pDAB9330 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,SzACS-2, and NoHetI. The pDAB9330 plasmid (FIG. 19; SEQ ID NO:45) wasconstructed using a multi-site Gateway L-R recombination reaction.pDAB9330 contains three PUFA synthase PTUs, one acyl-CoA synthetase PTU,one phosphopantetheinyl transferase PTU and a phosphinothricin acetyltransferase PTU. Specifically, the first PUFA synthase PTU contains thePvPhas Promoter v3, PvPhas 5′ UTR, SzPUFA OrfA v3, PvPhas 3′ UTR v1 andPvPhas 3′ MAR v2 (unannotated on the plasmid map). The second PUFAsynthase PTU contains the PvPhas Promoter v3, PvPhas 5′ UTR, SzPUFA OrfBv3, PvPhas 3′ UTR and PvPhas 3′ MAR v2 (unannotated on the plasmid map).The third PUFA synthase PTU contains the PvPhas Promoter v3, PvPhas 5′UTR, hSzThPUFA OrfC v3, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map). The acyl-CoA synthetase PTU containsthe PvPhas Promoter v3, PvPhas 5′ UTR, SzACS-2 v3 gene, PvPhas 3′ UTR v1and PvPhas 3′ MAR v2 (unannotated on the plasmid map). Thephosphopantetheinyl transferase PTU contains the PvPhas Promoter v3,PvPhas 5′ UTR, NoHetI v3, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map).

Plasmids pDAB9324, pDAB9325, pDAB9326, pDAB9329 and pDAB7333 wererecombined to form pDAB9330. Specifically, the five PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, SzACS-2 v3,NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the six PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Example 8.7 Construction of pDAB9337

pDAB9337 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,and NoHetI expression of which is driven by the phaseolin promoter. ThepDAB9337 plasmid (FIG. 20; SEQ ID NO:46) was constructed using amulti-site Gateway L-R recombination reaction.

pDAB9337 contains three PUFA synthase PTUs, one phosphopantetheinyltransferase PTU and a phosphinothricin acetyl transferase PTU.Specifically, the first PUFA synthase PTU contains the PvPhas Promoterv3, PvPhas 5′ UTR, SzPUFA OrfA v3, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map). The second PUFA synthase PTU containsthe PvPhas Promoter v3, PvPhas 5′ UTR, SzPUFA OrfB v3, PvPhas 3′ UTR v1and PvPhas 3′ MAR v2 (unannotated on the plasmid map). The third PUFAsynthase PTU contains the PvPhas Promoter v3, PvPhas 5′ UTR, hSzThPUFAOrfC v3, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2 (unannotated on theplasmid map). The phosphopantetheinyl transferase PTU contains thePvPhas Promoter v3, PvPhas 5′ UTR, NoHetI v3, PvPhas 3′ UTR v1 andPvPhas 3′ MAR v2 (unannotated on the plasmid map).

Plasmids pDAB9324, pDAB9325, pDAB9326, pDAB9328 and pDAB7333 wererecombined to form pDAB9337. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 8.8 Construction of pDAB9338

pDAB9338 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,and NoHetI. The phaseolin promoter is used to drive expression of SzPUFAOrfA, and PvDlec2 promoter is used to drive the other transgenes. ThepDAB9338 plasmid (FIG. 21; SEQ ID NO:47) was constructed using amulti-site Gateway L-R recombination reaction.

pDAB9338 contains three PUFA synthase PTUs, one phosphopantetheinyltransferase PTU and a phosphinothricin acetyl transferase PTU.Specifically, the first PUFA synthase PTU contains the PvPhas Promoterv3, PvPhas 5′ UTR, SzPUFA OrfA v3, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map). The second PUFA synthase PTU containsthe PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v3 and At2S SSPterminator v1. The third PUFA synthase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1. Thephosphopantetheinyl transferase PTU contains the PvDlec2 promoter v2, 2S5′ UTR, NoHetI v3 and At2S SSP terminator v1.

Plasmids pDAB9324, pDAB7335, pDAB7336, pDAB7338 and pDAB7333 wererecombined to form pDAB9338. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 8.9 Construction of pDAB9344

pDAB9344 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,and NoHetI all of which contain the Ribulose Bisphosphate Carboxylasesmall chain 1A (labeled as SSU-TP v1), which is fused to the aminoterminus of the coding sequence. The phaseolin promoter is used to driveexpression of SzPUFA OrfA, and PvDlec2 promoter is used to drive theother transgenes.

The pDAB9344 plasmid (FIG. 22; SEQ ID NO:48) was constructed using amulti-site Gateway L-R recombination reaction. pDAB9344 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvPhas Promoter v3, PvPhas 5′ UTR, SzPUFA OrfAv4, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2 (unannotated on the plasmidmap). The second PUFA synthase PTU contains the PvPhas Promoter v3,PvPhas 5′ UTR, SzPUFA OrfB v4, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map). The third PUFA synthase PTU containsthe PvPhas Promoter v3, PvPhas 5′ UTR, hSzThPUFA OrfC v4, PvPhas 3′ UTRv1 and PvPhas 3′ MAR v2 (unannotated on the plasmid map). Thephosphopantetheinyl transferase PTU contains the PvPhas Promoter v3,PvPhas 5′ UTR, NoHetI v4, PvPhas 3′ UTR v 1 and PvPhas 3′ MAR v2(unannotated on the plasmid map).

Plasmids pDAB9343, pDAB9342, pDAB9340, pDAB9331 and pDAB7333 wererecombined to form pDAB9344. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v4, SzPUFA OrfB v4, hSzThPUFA OrfC v4, NoHetI v4.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thesix PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 8.10 Construction of DAB9396

pDAB9396 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,SzACS-2, and NoHetI. The phaseolin promoter is used to drive expressionof SzPUFA OrfA and SzPUFA OrfB. The PvDlec2 promoter is used to drivethe other transgenes; hSzThPUFA OrfC, SzACS-2, and NoHetI.

The pDAB9396 plasmid (FIG. 23; SEQ ID NO:49) was constructed using amulti-site Gateway L-R recombination reaction. pDAB9396 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvPhas Promoter v3, PvPhas 5′ UTR, SzPUFA OrfAv3, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2 (unannotated on the plasmidmap). The second PUFA synthase PTU contains the PvDlec2 promoter v2, 2S5′ UTR, SzPUFA OrfB v3 and At2S SSP terminator v1. The third PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfCv3 and At2S SSP terminator v1. The acyl-CoA synthetase PTU contains thePvPhas Promoter v3, PvPhas 5′ UTR, SzACS-2 v3 gene, PvPhas 3′ UTR v1 andPvPhas 3′ MAR v2 (unannotated on the plasmid map). Thephosphopantetheinyl transferase PTU contains the PvDlec2 promoter v2, 2S5′ UTR, NoHetI v3 and At2S SSP terminator v1.

Plasmids pDAB9324, pDAB7335, pDAB7336, pDAB7339 and pDAB7333 wererecombined to form pDAB9338. Specifically, the five PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, SzACS-2 v3,NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the five PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Example 8.11 Construction of pDAB101412

pDAB101412 is a binary plasmid that was constructed to contain rebuilt,codon optimized versions of SzPUFA OrfA, SzPUFA OrfB, hSzThPUFA OrfC,SzACS-2, and NoHetI. The version of the phaseolin promoter used in thisconstruct was modified essentially as described in Bustos et al., 1989(The Plant Cell, Vol. 1; 839-853), wherein the 5′ portion of thepromoter was truncated and the phaseolin 5′ untranslated region was leftintact. The truncated phaseolin promoter sequences are identifiedthroughout this application as version 4 (v4), version 5 (v5), andversion 6 (v6). The pDAB101412 plasmid (FIG. 24; SEQ ID NO:50) wasconstructed using a multi-site Gateway L-R recombination reaction.

pDAB101412 contains three PUFA synthase PTUs, one acyl-CoA synthetasePTU, one phosphopantetheinyl transferase PTU and a phosphinothricinacetyl transferase PTU. Specifically, the first PUFA synthase PTUcontains the PvPhas Promoter v4, PvPhas 5′ UTR, SzPUFA OrfA v3 andAtuORF23 3′ UTR v1. The second PUFA synthase PTU contains the PvPhasPromoter v4, PvPhas 5′ UTR, SzPUFA OrfB v3 and AtuORF23 3′ UTR v1. Thethird PUFA synthase PTU contains the PvPhas Promoter v4, PvPhas 5′ UTR,hSzThPUFA OrfC v3 and AtuORF23 3′ UTR v1. The acyl-CoA synthetase PTUcontains the PvPhas Promoter v4, PvPhas 5′ UTR, 2S 5′ UTR, SzACS-2 v3gene and AtuORF23 5′ UTR v1. The phosphopantetheinyl transferase PTUcontains the PvPhas Promoter v5, PvPhas 5′ UTR, NoHetI v3 and AtuORF233′ UTR v1.

Plasmids pDAB7375, pDAB7376, pDAB7377, pDAB7398 and pDAB7333 wererecombined to form pDAB101412. Specifically, the five PTUs describedabove were placed in a head-to-tail orientation within the T-strand DNAborder regions of the plant transformation binary pDAB7333. The order ofthe genes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, SzACS-2v3, NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the five PTUs were then isolated and tested forincorporation of the six PTUs with restriction enzyme digestion and DNAsequencing.

Soybean Transformation with Promoters that Express Early in SeedDevelopment

The plasmids are used to stably transform soybean plants using theprotocols described above. Transgenic soybean plants are isolated andmolecularly characterized. The use of alternative constructs result insoybean plants that contain greater amounts of DHA and LC-PUFAs. Theresulting LC-PUFA accumulation is determined and soybean plants thatproduce 0.01% to 15% DHA or 0.01% to 15% LC-PUFA are identified.

Example 9 Expression of the Algal PUFA Synthase Gene Suite UsingAlternative Construct Designs Introducing Promoter Diversity to Reducethe Duplication of Regulatory Elements

Gene silencing is a phenomenon that has been observed in progenygenerations of transgenic soybean events. Several review articlesdiscuss Transcriptional Gene Silencing (TGS) and Post TranscriptionalGene Silencing (PTGS), such as those of Waterhouse I., 2001 (Nature411:834-842), Vaucheret and Fagard, 2001 (Trends in Genetics17(1):29-35, and Okamoto and Hirochika, 2001 (Trends in Plant Sci. 6(11): 527-534). In plants, gene silencing can be triggered by theduplication of transgenic polynucleotide sequences (tandem repeattransgene sequences, inverted repeat transgene sequences, or multipleinsertions into the chromosome) or when a sequence homologous to thetarget gene sequences is carried either by an infecting plant virus orby the T-DNA of Agrobacterium tumefaciens.

In addition, the duplication of transgene polynucleotide sequences canact as triggers for construct instability. Multiple transgene sequencesthat share high levels of sequence similarity can fold back on oneanother. Rearrangements can occur via homologous recombination, whereinintervening sequences of DNA are excised. As a result, fragments of DNAthat are located between repeated transgene polynucleotide sequences areexcised.

One strategy in designing plasmid vectors is to introduce promoterdiversity into a construct by incorporating multiple, unique seedspecific promoters that maintain high level expression of eachtransgene. Introducing promoter sequence diversity into the plasmidvectors can reduce gene silencing and improve plasmid stability.Multiple seed specific promoters include PvDlec2, Phaseolin, and Napin(U.S. Pat. No. 5,608,152). These promoters are relatively comparable inpromoter activity such as tissue specificity, levels of expression,duration of expression, etc.

Example 9.1 Construction of pDAB7733

The pDAB7733 binary plasmid (FIG. 25; SEQ ID NO:51) was constructedusing a multi-site Gateway L-R recombination reaction. pDAB7733 containsthree PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvPhas promoter v4, PvPhas 5′ UTR, SzPUFA OrfAv3 and AtuORF23 3′ UTR v1. The second PUFA synthase PTU contains theBnaNapinC promoter v1, BnaNapinC 5′ UTR, SzPUFA OrfB v3 and BnaNapinC 3′UTR v1. The third PUFA synthase PTU contains the PvDlec2 promoter v2, 2S5′ UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1. Thephosphopantetheinyl transferase PTU contains the PvPhas promoter v5,PvPhas 5′ UTR, NoHetI v3 and AtuOrf23 3′ UTR v1.

Plasmids pDAB7375, pDAB7731, pDAB7336, pDAB7378 and pDAB7333 wererecombined to form pDAB7733. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 9.2 Construction of pDAB7734

The pDAB7734 binary plasmid (FIG. 26; SEQ ID NO:52) was constructedusing a multi-site Gateway L-R recombination reaction. pDAB7734 containsthree PUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvPhas promoter v4, PvPhas 5′ UTR, SzPUFA OrfB v3 and AtuORF23 3′ UTRv1. The third PUFA synthase PTU contains the BnaNapinC promoter v1,BnaNapinC 5′ UTR, hSzThPUFA OrfC v3 and BnaNapinC 3′ UTR v1. Thephosphopantetheinyl transferase PTU contains the PvDlec2 promoter v2, 2S5′ UTR, NoHetI v3 and At2S SSP terminator v1.

Plasmids pDAB7334, pDAB7376, pDAB7732, pDAB7338 and pDAB7333 wererecombined to form pDAB7734. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the five PTUs with restriction enzyme digestion and DNA sequencing.

Example 9.3 Construction of pDAB 101493

The pDAB101493 binary plasmid (FIG. 27; SEQ ID NO:53) was constructedusing a multi-site Gateway L-R recombination reaction. pDAB101493contains three PUFA synthase PTUs, one phosphopantetheinyl transferasePTU and a phosphinothricin acetyl transferase PTU. Specifically, thefirst PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR,SzPUFA OrfA v3 and At2S SSP terminator v1. The second PUFA synthase PTUcontains the PvPhas promoter v4, PvPhas 5′ UTR, SzPUFA OrfB v3 andAtuORF23 3′ UTR v1. The third PUFA synthase PTU contains the PvDlec2promoter v2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1.The phosphopantetheinyl transferase PTU contains the PvPhas promoter v5,PvPhas 5′ UTR, NoHetI v3 and AtuOrf23 3′ UTR v1.

Plasmids pDAB7334, pDAB7376, pDAB7336, pDAB7378 and pDAB7333 wererecombined to form pDAB101493. Specifically, the four PTUs describedabove were placed in a head-to-tail orientation within the T-strand DNAborder regions of the plant transformation binary pDAB7333. The order ofthe genes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetIv3. pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 9.4 Construction of pDAB109507

The pDAB109507 plasmid (FIG. 28; SEQ ID NO:54) was constructed using amulti-site Gateway L-R recombination reaction. pDAB109507 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvPhas promoter v3, PvPhas 5′ UTR, SzPUFA OrfAv3 and PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2 (unannotated on the plasmidmap). The second PUFA synthase PTU contains the BnaNapinC promoter v1,BnaNapinC 5′ UTR, SzPUFA OrfB v3 and BnaNapinC 3′ UTR v1. The third PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfCv3 and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the BoACP promoter/5′ UTR v1, NoHetI v3 and AtuOrf23 3′ UTR v1.

Plasmids pDAB9324, pDAB7731, pDAB7336, pDAB101485 and pDAB7333 wererecombined to form pDAB109507. Specifically, the four PTUs describedabove were placed in a head-to-tail orientation within the T-strand DNAborder regions of the plant transformation binary pDAB7333. The order ofthe genes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetIv3. pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 9.5 Construction of pDAB 109508

The pDAB109508 plasmid (FIG. 29; SEQ ID NO:55) was constructed using amulti-site Gateway L-R recombination reaction. pDAB109508 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvPhas promoter v3, PvPhas 5′ UTR, SzPUFA OrfAv3 and PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2 (unannotated on the plasmidmap). The second PUFA synthase PTU contains the BnaNapinC promoter v1,BnaNapinC 5′ UTR, SzPUFA OrfB v3 and BnaNapinC 3′ UTR v1. The third PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfCv3 and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvDlec2 promoter v2, 2S 5′ UTR, NoHetI v3 and At2S SSPterminator v1.

Plasmids pDAB9324, pDAB7731, pDAB7336, pDAB7338 and pDAB7333 wererecombined to form pDAB109508. Specifically, the four PTUs describedabove were placed in a head-to-tail orientation within the T-strand DNAborder regions of the plant transformation binary pDAB7333. The order ofthe genes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetIv3. pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 9.6 Construction of pDAB 109509

The pDAB109509 plasmid (FIG. 30; SEQ ID NO:56) was constructed using amulti-site Gateway L-R recombination reaction. pDAB109509 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v3 and At2S SSP terminatorv1. The third PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1. Thephosphopantetheinyl transferase PTU contains the BoACP promoter/5′ UTRv1, NoHetI v3 and AtuOrf23 3′ UTR v1.

Plasmids pDAB7334, pDAB7335, pDAB7336, pDAB101485 and pDAB7333 wererecombined to form pDAB109509. Specifically, the four PTUs describedabove were placed in a head-to-tail orientation within the T-strand DNAborder regions of the plant transformation binary pDAB7333. The order ofthe genes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetIv3. pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Rearranging the Order of the Binary Construct PTUs to ReduceFragmentation of Long Gene Sequences

The SzPUFA OrfA PTU was placed at the 3′ end of the binary construct totest whether the order of the PTU cassettes could reduce fragmentationand rearrangements in isolated transgenic events. SzPUFA OrfA is a largeopen reading frame (˜8,700 b.p.) containing nine tandem acyl carrierprotein repeats. In the first series of completed constructs, the SzPUFAOrfA PTU was positioned to be integrated first into the plantchromosome. The SzPUFA OrfA PTU was subsequently followed by theremaining PUFA synthesis-related gene PTUs as they decreased inmolecular size. Molecular analysis of the SzPUFA OrfA coding regionindicated that some transgenic canola and Arabidopsis thaliana eventscontained fragmented insertions. Alternative Construct Designs aredescribed, wherein the order of the PUFA synthase PTUs has been changedto the following configuration; hSzThPUFA OrfC PTU, SzPUFA OrfB PTU,NoHetIPTU, SzPUFA OrfA PTU, and PAT PTU. Changing the location of theSzPUFA OrfA PTU on the binary construct is completed to reducefragmentation and rearrangement in isolated transgenic events.

Example 9.7 Construction of pDAB9151

The pDAB9151 plasmid (FIG. 31; SEQ ID NO:57) was constructed using amulti-site Gateway L-R recombination reaction. pDAB9151 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v3and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSPterminator v1. The phosphopantetheinyl transferase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, NoHetI v3 and At2S SSP terminator v1.The final PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR,SzPUFA OrfA v3 and At2S SSP terminator v1.

Plasmids pDAB9148, pDAB7335, pDAB9149, pDAB9150 and pDAB7333 wererecombined to form pDAB9151. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: hSzThPUFA OrfC v3, SzPUFA OrfB v3, NoHetI v3, SzPUFA OrfA v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Change the Transcriptional Direction of the Binary Construct PTUs toIntroduce Construct Diversity

An alternative construct design includes changing the order of PUFAsynthase PTUs and the transcriptional direction of the gene expressioncassettes. In the first series of completed constructs, each geneexpression cassette was positioned in the same direction (“head totail,” wherein the promoter of one gene expression cassette is locatedadjacent to the 3′ UTR of a second gene expression cassette). Thefollowing constructs describe a strategy wherein, gene expressioncassettes are positioned in different directions, and utilizealternative promoters. In these examples, the gene expression cassetteis located in trans to a second gene expression cassette such that thepromoters of both gene expression cassettes are engineered adjacent toone another. This configuration is described as a “head-to-head”configuration. Other configurations are described in the examples,wherein one gene expression cassettes is located in trans to a secondgene expression cassette such that the 3′ UTRs of both gene expressioncassettes are engineered adjacent to one another. This configuration isdescribed as a “tail-to-tail” configuration. To mitigate potentialread-through of such a design, the bidirectional Orf 23/24 terminatorhas been placed between these two PTUs. These configurations areproposed to increase expression of the transgenes, thereby resulting inhigher concentrations and content of LC-PUFA and DHA fatty acid.

Example 9.8 Construction of pDAB108207

The pDAB108207 plasmid (FIG. 32; SEQ ID NO:58) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108207 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvPhas promoter v6, PvPhas 5′ UTR, NoHetI v3, PvPhas 3′ UTRv1 and PvPhas 3′ MAR v2 (unannotated on the plasmid map). The secondPUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFAOrfC v3, At2S SSP terminator v1 and AtuORF23 3′ UTR v1. The third PUFAsynthase PTU contains the PvPhas promoter v6, PvPhas 5′ UTR, SzPUFA OrfBv3, PvPhas 3′ UTR and PvPhas 3′ MAR v2 (unannotated on the plasmid map)and AtuORF23 3′ UTR v1.

Plasmids pDAB7334, pDAB101489, pDAB108205, pDAB108206 and pDAB7333 wererecombined to form pDAB108207. Specifically, the SzPUFA OrfA v3 andNoHetI v3 are placed in a tail-to-tail orientation; NoHetI v3 andhSzThPUFA OrfC v3 are placed in a head-to-head orientation; hSzThPUFAOrfC v3 and SzPUFA OrfB are placed in a tail-to-tail orientation withinthe T-strand DNA border regions of the plant transformation binarypDAB7333. The order of the genes is: SzPUFA OrfA v3, NoHetI v3,hSzThPUFA OrfC v3, SzPUFA OrfB v3. pDAB7333 also contains thephosphinothricin acetyl transferase PTU: CsVMV promoter v2, PAT v5,AtuORF1 3′ UTR v4 in addition to other regulatory elements such asOverdrive and T-strand border sequences (T-DNA Border A and T-DNA BorderB). Recombinant plasmids containing the five PTUs were then isolated andtested for incorporation of the five PTUs with restriction enzymedigestion and DNA sequencing.

Example 9.9 Construction of pDAB108208

The pDAB108208 plasmid (FIG. 33; SEQ ID NO:59) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108208 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvPhas promoter v4, PvPhas 5′ UTR, NoHetI v3 and AtuORF233′ UTR v1. The second PUFA synthase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1. The thirdPUFA synthase PTU contains the PvPhas promoter v5, PvPhas 5′ UTR, SzPUFAOrfB v3, PvPhas 3′ UTR, PvPhas 3′ MAR v2 (unannotated on the plasmidmap), and AtuORF23 3′ UTR v1.

Plasmids pDAB108200, pDAB101490, pDAB108201, pDAB108202 and pDAB7333were recombined to form pDAB108208. Specifically, the SzPUFA OrfA v3 andNoHetI v3 are placed in a head-to-head orientation; NoHetI v3 andhSzThPUFA OrfC v3 are placed in a tail-to-tail orientation; hSzThPUFAOrfC v3 and SzPUFA OrfB are placed in a head-to-head orientation withinthe T-strand DNA border regions of the plant transformation binarypDAB7333. The order of the genes is: SzPUFA OrfA v3, NoHetI v3,hSzThPUFA OrfC v3, SzPUFA OrfB v3. pDAB7333 also contains thephosphinothricin acetyl transferase PTU: CsVMV promoter v2, PAT v5,AtuORF1 3′ UTR v4 in addition to other regulatory elements such asOverdrive and T-strand border sequences (T-DNA Border A and T-DNA BorderB). Recombinant plasmids containing the five PTUs were then isolated andtested for incorporation of the five PTUs with restriction enzymedigestion and DNA sequencing.

Example 9.10 Construction of pDAB108209

The pDAB108209 plasmid (FIG. 34; SEQ ID NO:60) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108209 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvPhas promoter v4, PvPhas 5′ UTR, NoHetI v3 and AtuORF233′ UTR v1. The second PUFA synthase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1. The thirdPUFA synthase PTU contains the PvPhas promoter v5, PvPhas 5′ UTR, SzPUFAOrfB v3, PvPhas 3′ UTR and PvPhas 3′ MAR v2 (unannotated on the plasmidmap), and random DNA spacer.

Plasmids pDAB108200, pDAB108204, pDAB108201, pDAB108202 and pDAB7333were recombined to form pDAB108209. Specifically, the SzPUFA OrfA v3 andNoHetI v3 are placed in a head-to-head orientation; NoHetI v3 andhSzThPUFA OrfC v3 are placed in a tail-to-tail orientation; hSzThPUFAOrfC v3 and SzPUFA OrfB are placed in a head-to-head orientation withinthe T-strand DNA border regions of the plant transformation binarypDAB7333. The order of the genes is: SzPUFA OrfA v3, NoHetI v3,hSzThPUFA OrfC v3, SzPUFA OrfB v3. pDAB7333 also contains thephosphinothricin acetyl transferase PTU: CsVMV promoter v2, PAT v5,AtuORF1 3′ UTR v4 in addition to other regulatory elements such asOverdrive and T-strand border sequences (T-DNA Border A and T-DNA BorderB). Recombinant plasmids containing the five PTUs were then isolated andtested for incorporation of the five PTUs with restriction enzymedigestion and DNA sequencing.

Doubling 3′ UTRs and Including Spacer DNA to Minimize TranscriptionalInterference.

Transcriptional interference can occur when multiple genes are stackedin a series thereby resulting in reduced expression of the downstreamgenes. This phenomenon results from transcriptional read -through of the3′ UTR and terminator into the next promoter-transcription unit.Alternative construct designs consisting of two strategies to minimizetranscriptional interference and transcriptional interference aredescribed. The first strategy deploys the use of two terminator/3′ UTRs,which are stacked between individual DHA gene expression cassettes tolimit read-through into the next gene expression cassette. The secondstrategy inserts about one-thousand base pairs of spacer DNA betweengene expression cassettes, thereby minimizing transcriptionalinterference.

Example 9.11 Construction of pDAB108207

The pDAB108207 plasmid (FIG. 32; SEQ ID NO:58) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108207 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The second PUFA synthase PTU contains thePvPhas promoter v3, PvPhas 5′ UTR, SzPUFA OrfB v3, PvPhas 3′ UTR, PvPhas3′ MAR v2 (unannotated on the plasmid map), and AtuORF23 3′ UTR v1. Thethird PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR,hSzThPUFA OrfC v3, At2S SSP terminator v1 and AtuORF23 3′ UTR v1. Thephosphopantetheinyl transferase PTU contains the PvPhas promoter v6,PvPhas 5′ UTR, NoHetI v3, PvPhas 3′ UTR v1 and PvPhas 3′ MAR v2(unannotated on the plasmid map).

Plasmids pDAB7334, pDAB101489, pDAB108205, pDAB108206 and pDAB7333 wererecombined to form pDAB108207. Specifically, the SzPUFA OrfA v3 andNoHetI v3 are placed in a tail-to-tail orientation and an AtuORF23 3′UTR is placed between the two PTUs; NoHetI v3 and hSzThPUFA OrfC v3 areplaced in a head-to-head orientation; hSzThPUFA OrfC v3 and SzPUFA OrfBare placed in a head-to-tail orientation and an AtuORF23 3′ UTR isplaced between the two PTUs within the T-strand DNA border regions ofthe plant transformation binary pDAB7333. The order of the genes is:SzPUFA OrfA v3, NoHetI v3, hSzThPUFA OrfC v3, SzPUFA OrfB v3. pDAB7333also contains the phosphinothricin acetyl transferase PTU: CsVMVpromoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to other regulatoryelements such as Overdrive and T-strand border sequences (T-DNA Border Aand T-DNA Border B). Recombinant plasmids containing the five PTUs werethen isolated and tested for incorporation of the five PTUs withrestriction enzyme digestion and DNA sequencing.

Example 9.12 Construction of pDAB108208

The pDAB108208 plasmid (FIG. 33; SEQ ID NO:59) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108208 contains threePUFA synthase PTUs, one acyl-CoA synthetase PTU, one phosphopantetheinyltransferase PTU and a phosphinothricin acetyl transferase PTU.Specifically, the first PUFA synthase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, SzPUFA OrfA v3 and At2S SSP terminator v1. The secondPUFA synthase PTU contains the PvPhas promoter v5, PvPhas 5′ UTR, SzPUFAOrfB v3, PvPhas 3′ UTR, PvPhas 3′ MAR v2 (unannotated on the plasmidmap) and AtuORF23 3′ UTR v1. The third PUPA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSPterminator v1. The phosphopantetheinyl transferase PTU contains thePvPhas promoter v4, PvPhas 5′ UTR, NoHetI v3 and AtuORF23 3′ UTR v1.

Plasmids pDAB108200, pDAB101490, pDAB108201, pDAB108202 and pDAB7333were recombined to form pDAB108208. Specifically, the SzPUFA OrfA v3 andNoHetI v3 are placed in a head-to-head orientation; NoHetI v3 andhSzThPUFA OrfC v3 are placed in a tail-to-tail orientation and anAtuORF23 3′ UTR is placed between the two PTUs; hSzThPUFA OrfC v3 andSzPUFA OrfB are placed in a head-to-head orientation within the T-strandDNA border regions of the plant transformation binary pDAB7333. Theorder of the genes is: SzPUFA OrfA v3, NoHetI v3, hSzThPUFA OrfC v3,SzPUFA OrfB v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the five PTUs were then isolated and tested forincorporation of the five PTUs with restriction enzyme digestion and DNAsequencing.

Example 9.13 Construction of pDAB108209

The pDAB108209 plasmid (FIG. 34; SEQ ID NO:60) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108209 contains threePUFA synthase PTUs, one acyl-CoA synthetase PTU, one phosphopantetheinyltransferase PTU and a phosphinothricin acetyl transferase PTU.Specifically, the first PUFA synthase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, SzPUFA OrfA v3 and At2S SSP terminator v1. The secondPUFA synthase PTU contains the PvPhas promoter v5, PvPhas 5′ UTR, SzPUFAOrfB v3, PvPhas 3′ UTR, PvPhas 3′ MAR v2 (unannotated on the plasmidmap), and random DNA spacer. The third PUFA synthase PTU contains thePvDlec2 promoter v2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSPterminator vl. The phosphopantetheinyl transferase PTU contains thePvPhas promoter v4, PvPhas 5′ UTR, NoHetI v3 and AtuORF23 3′ UTR v1.

Plasmids pDAB108200, pDAB108204, pDAB108201, pDAB108202 and pDAB7333were recombined to form pDAB108209. Specifically, the SzPUFA OrfA v3 andNoHetI v3 are placed in a head-to-head orientation; NoHetI v3 andhSzThPUFA OrfC v3 are placed in a tail-to-tail orientation and aone-thousand base pair spacer is placed between the two PTUs; hSzThPUFAOrfC v3 and SzPUFA OrfB are placed in a head-to-head orientation withinthe T-strand DNA border regions of the plant transformation binarypDAB7333. The order of the genes is: SzPUFA OrfA v3, NoHetI v3,hSzThPUFA OrfC v3, SzPUFA OrfB v3. pDAB7333 also contains thephosphinothricin acetyl transferase PTU: CsVMV promoter v2, PAT v5,AtuORF1 3′ UTR v4 in addition to other regulatory elements such asOverdrive and T-strand border sequences (T-DNA Border A and T-DNA BorderB). Recombinant plasmids containing the five PTUs were then isolated andtested for incorporation of the five PTUs with restriction enzymedigestion and DNA sequencing.

Using Alternative 3′ UTR-Terminator to Limit TranscriptionalRead-Through.

The Agrobacterium ORF 23 3′ UTR-terminator is primarily used toterminate transcription in many of the above constructs. It was recentlyshown the ZmLipase 3′ UTR-terminator is more effective in terminatingtranscriptional read-through in Arabidopsis thaliana. As such, oneversion of constructs utilizes the ZmLipase 3′ UTR-terminator incombination with the PvDlec2 promoter to test if this 3′ UTR can reducetranscriptional read-through of upstream genes, thereby reducingtranscriptional interference.

Example 9.14 Construction of pDAB9159

The pDAB9159 plasmid (FIG. 35; SEQ ID NO:61) was constructed using amulti-site Gateway L-R recombination reaction. pDAB9159 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and ZmLip 3′ UTR v1. The second PUFA synthase PTU contains the PvPhaspromoter v3, PvPhas 5′ UTR, SzPUFA OrfB v3 and ZmLip 3′ UTR v1. Thethird PUFA synthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR,hSzThPUFA OrfC v3 and ZmLip 3′ UTR v1. The phosphopantetheinyltransferase PTU contains the PvPhas promoter v3, PvPhas 5′ UTR, NoHetIv3 and ZmLip 3′ UTR v1.

Plasmids pDAB9152, pDAB9153, pDAB9154, pDAB9155 and pDAB7333 wererecombined to form pDAB9159. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Example 9.15 Construction of pDAB9147

The pDAB9147 plasmid (FIG. 36; SEQ ID NO:62) was constructed using amulti-site Gateway L-R recombination reaction. pDAB9147 contains threePUFA synthase PTUs, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfAv3, At2S SSP terminator v1 and ZmLip 3′ UTR v1. The second PUFA synthasePTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfB v3 and At2SSSP terminator v1. The third PUFA synthase PTU contains the PvDlec2promoter v2, 2S 5′ UTR, hSzThPUFA OrfC v3 and At2S SSP terminator v1.The phosphopantetheinyl transferase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, NoHetI v3 and At2S SSP terminator v1.

Plasmids pDAB9146, pDAB7335, pDAB7336, pDAB7338 and pDAB7333 wererecombined to form pDAB9147. Specifically, the four PTUs described abovewere placed in a head-to-tail orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfA v3, SzPUFA OrfB v3, hSzThPUFA OrfC v3, NoHetI v3.pDAB7333 also contains the phosphinothricin acetyl transferase PTU:CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 in addition to otherregulatory elements such as Overdrive and T-strand border sequences(T-DNA Border A and T-DNA Border B). Recombinant plasmids containing thefive PTUs were then isolated and tested for incorporation of the fivePTUs with restriction enzyme digestion and DNA sequencing.

Delivery of DHA Genes on Two Separate T-DNAs.

An alternative construct design consists of constructing two separatebinary vectors, the first vector containing a sub-set of PUFA synthasegenes on one T-DNA, and the second binary vector containing theremaining PUFA synthase genes on a second T-DNA. These binary vectorsare individually used to transform plants that are sexually crossed,thereby resulting in progeny that contain all of the PUFA synthase geneexpression constructs. An alternative method to produce transgenicplants would be to co-transform both binary vectors into soybean tissue,and select or screen for a single plant that contains both T-strands.

Example 9.16 Construction of pDAB108224

The pDAB108224 plasmid (FIG. 37; SEQ ID NO:63) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108224 contains onePUFA synthase PTU, one phosphopantetheinyl transferase PTU and aphosphinothricin acetyl transferase PTU. Specifically, the first PUFAsynthase PTU contains the PvDlec2 promoter v2, 2S 5′ UTR, SzPUFA OrfA v3and At2S SSP terminator v1. The phosphopantetheinyl transferase PTUcontains the PvPhas promoter v4, PvPhas 5′ UTR, NoHetI v3 and AtuORF233′ UTR v1.

Plasmids pDAB108216, pDAB108221 and pDAB7333 were recombined to formpDAB108224. Specifically, the SzPUFA OrfA v3 and NoHetI v3 are placed ina head-to-head orientation within the T-strand DNA border regions of theplant transformation binary pDAB7333. The order of the genes is: SzPUFAOrfA v3, NoHetI v3. pDAB7333 also contains the phosphinothricin acetyltransferase PTU: CsVMV promoter v2, PAT v5, AtuORF1 3′ UTR v4 inaddition to other regulatory elements such as Overdrive and T-strandborder sequences (T-DNA Border A and T-DNA Border B). Recombinantplasmids containing the five PTUs were then isolated and tested forincorporation of the three PTUs with restriction enzyme digestion andDNA sequencing.

Example 9.17 Construction of pDAB108225

The pDAB108225 plasmid (FIG. 38; SEQ ID NO:64) was constructed using amulti-site Gateway L-R recombination reaction. pDAB108225 contains twoPUFA synthase PTUs and a phosphinothricin acetyl transferase PTU.Specifically, the first PUFA synthase PTU contains the PvDlec2 promoterv2, 2S 5′ UTR, SzPUFA OrfB v3 and At2S SSP terminator v1. The secondPUFA synthase PTU contains the PvPhas promoter v4, SzPUFA OrfB v3 andAtu ORF23 3′ UTR v1.

Plasmids pDAB108217, pDAB108222 and pDAB7333 were recombined to formpDAB108225. Specifically, the SzPUFA (MB v3 and hSzThPUFA OrfC v3 areplaced in a head-to-head orientation within the T-strand DNA borderregions of the plant transformation binary pDAB7333. The order of thegenes is: SzPUFA OrfB v3, hSzThPUFA OrfC v3. pDAB7333 also contains thephosphinothricin acetyl transferase PTU: CsVMV promoter v2, PAT v5,AtuORF1 3′ UTR v4 in addition to other regulatory elements such asOverdrive and T-strand border sequences (T-DNA Border A and T-DNA BorderB). Recombinant plasmids containing the five PTUs were then isolated andtested for incorporation of the three PTUs with restriction enzymedigestion and DNA sequencing.

Soybean Transformation with Constructs Containing Alternative Designs

These plasmids are used to stably transform soybean plants using theprotocols described above. Transgenic soybean plants are isolated andmolecularly characterized. The use of alternative constructs result insoybean plants that contain greater amounts of DHA and LC-PUFAs. Theresulting LC-PUFA accumulation is determined and soybean plants thatproduce 0.01% to 15% DHA or 0.01% to 15% LC-PUFA are identified.

Example 10 Alternative Construct Designs Used for Transformation ofArabidopsis thaliana and Subsequent Production of LC-PUFA and DHA

Arabidopsis thaliana plants were transformed with Agrobacteriumtumefaciens strains containing the pDAB101493, pDAB7362, pDAB7369,pDAB101412, or pDAB7380 binary vectors. A floral dipping transformationprotocol described by Clough and Bent (1998) was used for thetransformation. Clough and Bent, “Floral dip: a simplified method foragrobacterium-mediated transformation of Arabidopsis thalia,” Plant J.,16:735-743, 1998. Transformed Arabidopsis plants were obtained andmolecular confirmation of the transgene presence was completed. Tiplants from the transgenic Arabidopsis events were grown to maturity inthe greenhouse. These plants were self-fertilized and the resulting T₂seed harvested at maturity, T₂ seeds (10 mg) were analyzed via FAMEsGC-FID to determine the LC-PUFA and DHA content in the T₂ Arabidopsisseed. The tissue was analyzed via the FAMEs GC-FID method as describedin the previous examples. T₂ seeds from a T₁ plant of the Arabidopsisplants contained from 0% to 0.95% DHA and 0% to 1.50% total LC-PUFA. TheLC-PUFA and DHA content of the T₂ seed from individual T₁ plants isshown in FIG. 39.

Example 11 Co-Expression of DGAT2 or ACCase with the Algal PUFA SynthaseGene Suite within Soybean

Oil content within soybean plants is further modified by transformationof chimeric DNA molecules that encode and express an acetyl CoAcarboxylase (ACCase) or a type 2 diacylglycerol acyltransferase (DGAT2).These genes are co-expressed with the algal PUFA synthase genesdescribed above, either through breeding soybean plants containing theACCase or DGAT2 expression cassette with soybean plants containing thePUFA synthase genes; or by transforming soybean plants with a gene stackcontaining the ACCase or DGAT2 and the PUFA synthase genes. Regulatoryelements necessary for expression of an ACCase or DGAT2 coding sequencecan include those described above. Additional regulatory elementsexpression sequences known in the art may also be used. The ACCase andDGAT2 expression cassettes are transformed into soybean usingtransformation protocols described above. Transformation may occur asmolecular stacks of the ACCase or DGAT2 expression cassette combinedwith the PUFA synthase OrfA, PUFA synthase OrfB, PUFA synthase OrfC,acyl-CoA synthetase and 4′ phosphopantetheinyl transferase HetIexpression cassettes; or as independent ACCase or DGAT2 expressioncassettes linked to a selectable marker and then subsequently crossedwith soybean plants that contain the PUFA synthase OrfA, PUFA synthaseOrfB, PUFA synthase OrfC, acyl-CoA synthetase and 4′ phosphopantetheinyltransferase HetI expression cassettes. Positive transformants areisolated and molecularly characterized. Soybean plants are identifiedthat contain increased accumulation of LC-PUFAs in the plant, the seedof the plant, or plant oil concentrations compared to untransformedcontrol soybean plants. Such increases can range from a 1.2 to a 20-foldincrease.

The over-expression of ACCase in the cytoplasm may produce higher levelsof malonyl-CoA, Soybean plants or seed containing increased levels ofcytoplasmic malonyl-CoA may produce subsequently higher levels of thelong-chain polyunsaturated fatty acid (LC-PUFA) when the algal PUFAsynthase genes are present and expressed. DGAT2 genes that are expressedwithin soybean plants may be capable of preferentially incorporatingsignificant amounts of docosahexaenoic acid (DHA) and eicosapentaenoicacid (EPA) into triacylglycerol. DGAT2 genes with substrate preferencetoward LC-PUFAs (see, e.g., PCT International Publication WO 2009/085169A2) may increase incorporation of these fatty acids into triacylglycerol(TAG). Such DGAT genes are useful for directing the incorporation ofLC-PUFA, particularly DHA, into TAG and for increasing the production ofTAG in plants and other organisms.

Example 12 Production of DHA in Arabidopsis Seeds Transformed withAlternative Construct Designs for Expression of PUFA Synthase Genes

Arabidopsis T₁ events transformed with Agrobacterium tumefaciensharboring plasmids encoding PUFA synthase genes and HetI (and in somecases SzACS-2) under the control of various plant expression elementswere generated using the floral dip method essentially as described inClough and Bent (Plant J., 1998 16(6):735-43). The resulting Ti seed washarvested and sown. Transformed Ti plants were selected by spraying withphosphinothricin to select for those plants containing a functional PATgene as a selectable marker. Leaf tissue from the surviving T₁ plantswas sampled and analyzed by quantitative PCR reactions specific for thePAT gene to identify those plants containing a single copy of theselectable marker (and associated transgenes). These plants were grownto maturity, the T₂ seed harvested and analyzed for LC-PUFA content (as% of total extractable FAMEs). A summary of data from the eventsgenerated with various constructs encoding PUFA synthase genes is shownin Table 12.

TABLE 12 Arabidopsis events containing a single copy of the PATtransgene producing LC-PUFA in T₂ seeds and the levels of DHA and EPAfor each event, shown as a percentage of total Oil. # of # of events #of events with Average Maximum Maximum Maximum Average events producingLC-PUFA LC-PUFA LC-PUFA DHA EPA n-3/PUFA Construct generated LC-PUFA >1%¹ content ² content content ³ content ⁴ ratio ⁵ pDAB9167 30  9 (30%)  00.06 0.24 0.17 0 67% pDAB101477 11  2 (18%)  0 0.07 0.49 0.29 0 64%pDAB101412 63 33 (52%)  0 0.17 0.91 0.40 0.07 68% pDAB7380 45 23 (51%) 0 0.23 0.79 0.47 0.12 69% pDAB7733 23 13 (57%)  0 0.24 1.07 0.69 0.0761% pDAB101493 25 15 (60%)  0 0.26 0.88 0.52 0.13 75% pDAB100518 71 39(71%)  0 0.27 0.96 0.64 0.07 70% pDAB7362 126 45 (36%) 10 (8%) 0.28 1.731.02 0.26 64% pDAB9151 35 15 (43%)  3 (9%) 0.29 1.39 0.84 0.11 74%pDAB9147 40 19 (48%)  3 (8%) 0.36 1.62 0.89 0.10 70% pDAB9159 46 32(70%)  0 0.43 1.07 0.68 0.13 72% pDAB109509 32 21 (66%)  1 (3%) 0.441.14 0.79 0.17 67% pDAB7734 45 27 (60%)  8 (18%) 0.49 1.62 1.00 0.13 76%pDAB7369 42 26 (62%)  5 (12%) 0.50 1.47 0.88 0.11 66% pDAB108209 46 36(78%)  2 (4%) 0.62 1.61 1.01 0.29 70% pDAB109508 29 20 (69%)  7 (24%)0.68 1.72 1.02 0.13 64% pDAB108208 46 33 (72%) 21 (46%) 0.71 1.33 0.890.18 73% pDAB109507 30 23 (77%) 10 (33%) 0.77 2.03 1.45 0.05 72%pDAB108207 47 35 (74%) 16 (34%) 0.86 1.82 0.99 0.16 64% ¹ Number ofevents with LC-PUFA content >1% of total seed FAMEs with %-age of totalevents in parentheses. ² Average total LC-PUFA content (DHA(n-3) +EPA(n-3) + DPA (n-6)) of all T2 seed samples as % of total seed FAMEs ³Maximum DHA content of all T₂ seed samples analyzed as % of total FAMEs⁴ Maximum EPA content of all T₂ seed samples analyzed as % of totalFAMEs ⁵ Average n-3 LC-PUFA (DHA + EPA)/Total LC-PUFA content across allLC-PUFA-producing events (as %)

These data show that certain construct configurations and promotercombinations generate a higher proportion of events containing LC-PUFAin the T₂ seed (77% of all single copy events for pDAB109507 producedDHA, and 86% of all single copy events for pDAB108207 produced DHA).Also certain constructs generate a higher proportion of eventsproducing >1% LC-PUFA content (33% of all single copy events forpDAB109507, and 34% of all single copy events for pDAB108207). Themaximum LC-PUFA content of the T₂ seed from the various events rangedfrom 0.24%-2.03% for the different constructs. Likewise, certainconstructs produce higher levels of omega-3 LC-PUFAs. The maximum DHAcontent ranged from 0.17%-1.45% and the maximum EPA content ranged from0%-0.26% across all the constructs and events generated. These dataindicate that the alteration of the construct design where promoterconfigurations were changed resulted in transgenic plants that exhibitincreased LC-PUFA, as compared to transgenic plants that weretransformed with pDAB7362. As such, these constructs in which theconstruct design was altered are desirable for crop transformations.

T₂ seed from high LC-PUFA producing events was planted and the leaftissue from the growing plants was sampled using quantitative PCR toassay the PAT gene and other transgenes. Plants containing two copies ofthe transgenes (i.e., homozygotes) were identified and grown tomaturity. The resulting T₃ seed was harvested and analyzed for LC-PUFAcontent. Some constructs such as pDAB7362 and pDAB109509, whichcontained repeated promoter/3′ UTR expression elements, showed poorstability of the LC-PUFA trait in the subsequent T₃ seed generation.However, some events transformed with different construct configurationsand/or diversified expression elements (e.g., pDAB108207, 109508 and7734) produced significantly improved stability of the LC-PUFA traitinto the T₃ seed generation, as shown in Table 13. These data indicatethat certain constructs can maintain stability of the DHA trait insubsequent generations and that such constructs are preferred for croptransformations.

TABLE 13 LC-PUFA analysis of T₃ seed progeny from selected transgenicArabidopsis DHA-producing T₂ lines Parent Parent T₂ seed No. of AverageRange of Average Range of T₃ T₂ seed LC-PU homozygous T₃ seed T₃ seed T₃seed seed DHA FA progeny DHA DHA LC-PUFA LC-PUFA Construct Event IDcontent content analyzed content ¹ content content content pDAB73625217[12]- 0.66 1.53 14 0.03   0-0.10 0.17   0-0.46 202 pDAB73625217[12]- 0.89 1.50 20 0.04   0-0.28 0.08   0-0.48 231 pDAB73625217[12]- 0.77 1.35 19 0.03   0-0.17 0.05   0-0.26 219 pDAB109509109509[1]- 0.79 1.14 10 0.13   0-0.31 0.20   0-0.42 025 pDAB109509109509[1]- 0.61 1.00 10 0.15 0.06-0.30 0.21 0.09-0.42 037 pDAB1095091095092[2]- 0.73 1.03 10 0.09   0-0.36 0.12   0-0.47 102 pDAB108207108207[1]- 0.93 1.57 10 0.89 0.66-1.09 1.43 0.99-1.84 047 pDAB108207108207[1]- 0.99 1.77 5 1.08 0.99-1.27 2.05 1.83-2.36 051 pDAB108207108207[1]- 0.97 1.68 5 0.88 0.55-1.04 1.64 1.08-1.9  076 pDAB109508109508[1]- 1.02 1.72 10 1.25 1.16-1.39 1.99 1.86-2.09 028 pDAB7734649[1]- 1 1.62 9 1.43 0.98-1.83 2.17 1.45-2.89 138 Total LC-PUFAcontents and DHA contents are % of total FAMEs ¹ T₃ bulk seed from 5-20individual homozygous plants was analyzed

The foregoing description of the invention has been presented forpurposes of illustration and description. Furthermore, the descriptionis not intended to limit the invention to the form disclosed herein.

All of the various aspects, embodiments, and options described hereincan be combined in any and all variations.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A method of genetically modifying a soybeanplant, descendant, cell, tissue, seed or part thereof, comprisingmodifying a soybean plant to include: (i) a nucleic acid moleculecomprising a soybean codon-optimized coding sequence encoding an algalpolyunsaturated fatty acid (PUFA) synthase that produces at least onePUFA; and (ii) a nucleic acid molecule comprising a soybeancodon-optimized coding sequence encoding algal phosphopantetheinyltransferase (PPTase).
 2. The method of claim 1, wherein the PUFAsynthase and the PPTase enzymes are from Schizochytrium orThraustochytrium microalgae.
 3. The method of claim 1, wherein the PUFAsynthase comprises an amino acid sequence that is at least 80% identicalto the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
 4. The method of claim 1, whereinthe PUFA synthase comprises the amino acid sequence of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
 5. Themethod of claim 1, wherein the PUFA synthase comprises the amino acidsequences of SEQ ID NOs:1-3 or SEQ ID NOs:6-8.
 6. The method of claim 1,wherein the PPTase comprises an amino acid sequence that is at least 80%identical to SEQ ID NO:5 or SEQ ID NO:
 10. 7. The method of claim 1,wherein the PPTase comprises the amino acid sequence of SEQ ID NO:5 orSEQ ID NO:
 10. 8. The method of claim 3, wherein the PPTase comprises anamino acid sequence that is at least 80% identical to SEQ ID NO:5 or SEQID NO:
 10. 9. The method of claim 4, wherein the PPTase comprises theamino acid sequence of SEQ ID NO:5 or SEQ ID NO: 10
 10. The method ofclaim 1, wherein the nucleic acid sequences of (i) and (ii) arecontained in a single recombinant expression vector.
 11. The method ofclaim 1, wherein the nucleic acid sequence of (i) or (ii) is operablylinked to a seed-specific promoter or a leaf-specific promoter.
 12. Themethod of claim 1, wherein the nucleic acid sequence of (i) or (ii) isoperably linked to PvDlec2, LfKCS3, FAE 1, BoACP, BnaNapinC, ubiquitinor CsVMV promoter.
 13. The method of claim 1, further comprisingmodifying the soybean plant to include: (iii) a nucleic acid moleculecomprising a nucleic acid sequence encoding an acyl-CoA synthetase(ACoAS).
 14. The method of claim 13, wherein the ACoAS comprises anamino acid sequence that is at least 80% identical to SEQ ID NO:4 or SEQID NO:9.
 15. The method of claim 13, wherein the ACoAS comprises theamino acid sequence of SEQ ID NO:4 or SEQ ID NO:9.
 16. The method ofclaim 13, wherein the nucleic acid sequence of (iii) is operably linkedto a seed-specific promoter or a leaf-specific promoter.
 17. The methodof claim 13, wherein the nucleic acid sequence of (iii) is operablylinked to PvDlec2, LfKCS3, FAE 1, BoACP, BnaNapinC, ubiquitin or CsVMVpromoter.
 18. The method of claim 13, wherein the nucleic acid sequenceof (iii) encodes an acetyl CoA carboxylase (ACCase) or a type 2diacylglycerol acyltransferase (DGAT2).